Pharmaceutical compositions of 1,2,4-oxadiazole benzoic acid and their use for the treatment of disease

ABSTRACT

Novel 1,2,4-oxadiazole benzoic acid compounds, methods of using and pharmaceutical compositions comprising an 1,2,4-oxadiazole benzoic acid derivative are disclosed. The methods include methods of treating or preventing a disease ameliorated by modulation of premature translation termination or nonsense-mediated mRNA decay, or ameliorating one or more symptoms associated therewith.

This application is a continuation of U.S. application Ser. No.11/042,652, filed Jan. 24, 2005, now U.S. Pat No. 7,772,259 currentlyallowed, which is a division of U.S. application Ser. No. 10/822,259,filed Apr. 9, 2004, now U.S. Pat. No. 6,992,096 B2, issued Jan. 31,2006, which claims the benefit of U.S. Provisional Application No.60/461,988, filed Apr. 11, 2003, the contents of each of which areincorporated by reference herein in its entirety.

1. FIELD OF INVENTION

The invention relates to 1,2,4-oxadiazole benzoic acid compounds,compositions comprising the compounds and methods for treating orpreventing diseases associated with nonsense mutations of mRNA byadministering these compounds or compositions.

2. BACKGROUND OF THE INVENTION

Gene expression in cells depends upon the sequential processes oftranscription and translation. Together, these processes produce aprotein from the nucleotide sequence of its corresponding gene.

Transcription involves the synthesis of mRNA from DNA by RNA polymerase.Transcription begins at a promoter region of the gene and continuesuntil termination is induced, such as by the formation of a stem-loopstructure in the nascent RNA or the binding of the rho gene product.

Protein is then produced from mRNA by the process of translation,occurring on the ribosome with the aid of tRNA, tRNA synthetases andvarious other protein and RNA species. Translation comprises the threephases of initiation, elongation and termination. Translation isinitiated by the formation of an initiation complex consisting ofprotein factors, mRNA, tRNA, cofactors and the ribosomal subunits thatrecognize signals on the mRNA that direct the translation machinery tobegin translation on the mRNA.

Once the initiation complex is formed, growth of the polypeptide chainoccurs by the repetitive addition of amino acids by the peptidyltransferase activity of the ribosome as well as tRNA and tRNAsynthetases. The presence of one of the three termination codons (UAA,UAG, UGA) in the A site of the ribosome signals the polypeptide chainrelease factors (RFs) to bind and recognize the termination signal.Subsequently, the ester bond between the 3′ nucleotide of the tRNAlocated in the ribosome's P site and the nascent polypeptide chain ishydrolyzed. The completed polypeptide chain is released, and theribosome subunits are recycled for another round of translation.

Mutations of the DNA sequence in which the number of bases is alteredare categorized as insertion or deletion mutations (frameshiftmutations) and can result in major disruptions of the genome. Mutationsof the DNA that change one base into another are labeled missensemutations and are subdivided into the classes of transitions (one purineto another purine, or one pyrimidine to another pyrimidine) andtransversions (a purine to a pyrimidine, or a pyrimidine to a purine).

Insertions, deletions, transition and transversion mutations can allresult in a nonsense mutation, or chain termination mutation, in whichthe base mutation or frameshift mutation changes an amino acid codoninto one of the three stop codons. These premature stop codons canproduce aberrant proteins in cells as a result of premature translationtermination. A nonsense mutation in an essential gene can be lethal andcan also result in a number of diseases, such as, cancers, lysosomalstorage disorders, the muscular dystrophies, cystic fibrosis andhemophilia, to name a few.

In bacterial and eukaryotic strains with nonsense mutations, suppressionof the nonsense mutation can arise as a result of a mutation in one ofthe tRNA molecules so that the mutant tRNA can recognize the nonsensecodon, as a result of mutations in proteins that are involved in thetranslation process, as a result of mutations in the ribosome (eitherthe ribosomal RNA or ribosomal proteins), or by the addition ofcompounds known to alter the translation process (for example,cycloheximide or the aminoglycoside antibiotics). The result is that anamino acid will be incorporated into the polypeptide chain at the siteof the nonsense mutation, and translation will not prematurely terminateat the nonsense codon. The inserted amino acid will not necessarily beidentical to the original amino acid of the wild-type protein; however,many amino acid substitutions do not have a gross effect on proteinstructure or function. Thus, a protein produced by the suppression of anonsense mutation would be likely to possess activity close to that ofthe wild-type protein. This scenario provides an opportunity to treatdiseases associated with nonsense mutations by avoiding prematuretermination of translation through suppression of the nonsense mutation.

The ability of aminoglycoside antibiotics to promote readthrough ofeukaryotic stop codons has attracted interest in these drugs aspotential therapeutic agents in human diseases caused by nonsensemutations. One disease for which such a therapeutic strategy may beviable is classical late infantile neuronal ceroid lipofuscinosis(LINCL), a fatal childhood neurodegenerative disease with currently noeffective treatment. Premature stop codon mutations in the gene CLN2,encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I), are associatedwith disease in approximately half of children diagnosed with LINCL. Theability of the aminoglycoside gentamicin to restore TPP-I activity inLINCL cell lines has been examined. In one patient-derived cell linethat was compound heterozygous for a commonly seen nonsense mutation(Arg208Stop) and a different rare nonsense mutation, approximately 7% ofnormal levels of TPP-I were maximally restored with gentamicintreatment. These results suggest that pharmacological suppression ofnonsense mutations by aminoglycosides or functionally similarpharmaceuticals may have therapeutic potential in LINCL (Sleat et. al.,Eur. J. Ped. Neurol. 5:Suppl A 57-62 (2001)).

In cultured cells having premature stop codons in the Cystic FibrosisTransmembrane Conductance Regulator (CFTR) gene, treatment withaminoglycosides led to the production of full length CFTR (Bedwell et.al., Nat. Med. 3:1280-1284 (1997); Howard et. al. Nat. Med. 2: 467-469(1996)). In a mouse model for Duchenne muscular dystrophy, gentamicinsulfate was observed to suppress translational termination at apremature stop codon resulting in full length dystrophin (Barton-Daviset. al., J. Clin. Invest. 104:375-381 (1999)). A small increase in theamount of full length dystrophin provided protection againstcontraction-induced damage in the mdx mice. The amino acid inserted atthe site of the nonsense codon was not determined in these studies.

Small molecule therapeutics or prophylactics that suppress prematuretranslation termination by mediating the misreading of the nonsensecodon would be useful for the treatment of a number of diseases. Thediscovery of small molecule drugs, particularly orally bioavailabledrugs, may lead to the introduction of a broad spectrum of selectivetherapeutics which can be used against disease caused by nonsensemutations.

3. SUMMARY OF THE INVENTION

The invention encompasses novel compounds, novel pharmaceuticalcompositions and novel methods of treatment. The compounds,compositions, and methods are, in part, based upon the modulation ofpremature translation termination and/or nonsense-mediated mRNA decaythat play a role in a variety of diseases. Such diseases can occur dueto the decreased amount of active protein produced as a result ofpremature termination of translation. The compounds of the inventionallow the translation of mRNA to continue past the nonsense mutationresulting in the production of full length protein. Thus the inventionencompasses compounds, compositions, and methods for treating andpreventing a variety of diseases, in particular genetic diseases.

This invention encompasses 1,2,4-oxadiazole benzoic acid compounds offormula I:

or pharmaceutically acceptable salts, hydrates, clathrates, prodrugs,polymorphs, stereoisomers, including enantiomers, diastereomers,racemates or mixtures of stereoisomers, thereof wherein:

Z is substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted heterocycle, substituted or unsubstituted arylalkyl;

R¹ is hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, —(CH₂CH₂O)_(n)R⁶ or any biohydrolyzable group;

R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl; substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy,aryloxy, heteroaryloxy, halogen, CF₃, OCF₃, OCHF₂, CN, COOH, COOR⁷,SO₂R⁷, NO₂, NH₂, or N(R⁷)₂;

each occurrence of R⁷ is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl; substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy,aryloxy, heteroaryloxy, halogen or CF₃; and

n is an integer from 1 to 7.

In a related embodiment, the invention encompasses 1,2,4-oxadiazolebenzoic acid compounds of the formula II:

or pharmaceutically acceptable salts, hydrates, clathrates, orstereoisomers thereof wherein Z is defined as in formula I and R ishydrogen or halogen.

In a preferred embodiment of the invention, the compounds of formulas Iand II are pharmaceutically acceptable salts, hydrates, clathrates,prodrugs, polymorphs, bio-hydrolyzable esters, racemates, or purifiedstereoisomers including, but not limited to, optically pure enantiomersand diastereomers.

The invention further encompasses methods of treating or preventing adisease ameliorated by modulation of premature translation terminationor nonsense-mediated mRNA decay, or ameliorating one or more symptomsassociated therewith comprising administering to a patient in needthereof a therapeutically or prophylactically effective amount of acompound of the formula I or II and pharmaceutically acceptable salts,hydrates, solvates, clathrates, prodrugs or polymorphs thereof. In apreferred embodiment, the disease is a genetic disease; a CNS disease;an inflammatory disease; a neurodegenerative disease; an autoimmunedisease; a proliferative disease, in particular cancer; a cardiovasculardisease; or a pulmonary disease; more preferably the disease includes,but is not limited to, amyloidosis, LINCL, hemophilia, Alzheimer'sdisease, atherosclerosis, giantism, dwarfism, hypothyroidism,hyperthyroidism, cystic fibrosis, aging, obesity, Parkinson's disease,Niemann Pick's disease, cystic fibrosis, familial hypercholesterolemia,retinitis pigmentosa, Duchenne muscular dystrophy, or Marfan syndrome.

The invention further encompasses methods of treating or preventing, orameliorating a genetic disease one or more symptoms associated with ormanifestations of a genetic disease comprising administering to apatient in need thereof a therapeutically or prophylactically effectiveamount of a compound of the formula I or II and pharmaceuticallyacceptable salts, hydrates, solvates, clathrates, prodrugs or polymorphsthereof. In a preferred embodiment, the disease is a CNS disease; aninflammatory disease; a neurodegenerative disease; a cardiovasculardisease; an autoimmune disease; cancer; more preferably, the geneticdisease includes, but is not limited to, amyloidosis, LINCL, hemophilia,Alzheimer's disease, atherosclerosis, giantism, dwarfism,hypothyroidism, hyperthyroidism, cystic fibrosis, aging, obesity,Parkinson's disease, Niemann Pick's disease, cystic fibrosis, familialhypercholesterolemia, retinitis pigmentosa, Duchenne muscular dystrophy,or Marfan syndrome.

The invention further relates to methods of treating, preventing, orameliorating cancer or one or more symptoms associated with ormanifestations of cancer comprising administering to a patient in needthereof a therapeutically or prophylactically effective amount of acompound of the formula I or H and pharmaceutically acceptable salts,hydrates, solvates, clathrates, prodrugs or polymorphs thereof.

In a preferred embodiment of the invention, the patient is a mammal,more preferably a human susceptible to or at risk of acquiring a geneticdisease. In an alternative embodiment, the patient has undergone ascreening process to determine the presence of a nonsense mutationcomprising the steps of screening a subject or cells extracted therefromby an acceptable nonsense mutation screening assay. In a relatedembodiment, the therapy is personalized in that the patient is screenedfor a nonsense mutation screening assay and treated by theadministration of one or more compounds of the invention; particularly,the patient may be treated with a compound particularly suited for themutations in question, e.g., depending upon the disease type, cell type,and the gene in question. In a further embodiment, the patient is aninfant or child. In yet another embodiment, the invention encompassesthe treatment of pregnant woman or the fetus directly.

In a still preferred embodiment of the invention, the compound isadministered parenterally, transdermally, mucosally, nasally, buccally,sublingually, or orally; more preferably the compound is administeredorally, most preferably the compound is administered orally in the formof a tablet, capsule or liquid.

The invention encompasses methods for modulating premature translationtermination and/or nonsense-mediated mRNA decay. The invention furtherencompasses a method for suppressing premature translation terminationand/or nonsense-mediated mRNA decay in a cell comprising contacting acell exhibiting premature translation termination and/ornonsense-mediated mRNA decay with an effective amount of a compound offormula I or II. The invention further encompasses a method for inducingnonsense suppression in a cell comprising contacting a cell exhibiting anonsense mutation with an effective amount of a compound of formula I orII. A nonsense codon can be present in the DNA or RNA of any type ofcell and can arise naturally or result from mutagenesis. Accordingly,cells encompassed by the present methods include animal cells, mammaliancells, bacterial cells, plant cells and virally infected cells. In oneembodiment, the nonsense codon was present in the progenitor DNA. Inanother embodiment, the nonsense codon resulted from mutagenesis.

Without being limited to any particular theory, the ability of thecompounds of formula I or II to promote readthrough of stop codons makesthem useful in the treatment or prevention of any disease which iscaused in whole or in part by a nonsense mutation. Such diseases canoccur due to the decreased amount of active protein produced as a resultof premature termination of translation. Without being limited to anyparticular theory, the compounds of formula I or II allow thetranslation of mRNA to continue past the nonsense mutation resulting inthe production of full length protein. A powerful aspect of theinvention is that the therapeutic activity of compounds of formula I orII are not necessarily disease specific, instead are effective attreating of preventing any disease associated with a nonsense mutation.Further, the methods of the invention may be patient specific. That is,a patient may be screened to determine if this disease is associatedwith a nonsense mutation. If so, they can be treated with a compound ofthe invention.

The compounds of formula I or II are useful for treating or preventinggenetic diseases. Genetic diseases that can be treated or prevented bycompounds of formula I or II include cancer, autoimmune disease, blooddisease, collagen disease, diabetes, inflammatory diseases or a centralnervous system disease.

3.1 Definitions

As used herein, “premature translation termination” refers to the resultof a mutation that changes a codon corresponding to an amino acid to astop codon.

As used herein, “nonsense-mediated mRNA decay” refers to any mechanismthat mediates the decay of mRNAs containing a premature translationtermination codon.

As used herein, a “premature termination codon” or “premature stopcodon” refers to the occurrence of a stop codon where a codoncorresponding to an amino acid should be.

As used herein, a “nonsense mutation” is a point mutation changing acodon corresponding to an amino acid to a stop codon.

As used herein, “nonsense suppression” refers to the inhibition orsuppression of premature translation and/or nonsense-mediated mRNAdecay.

As used herein, “modulation of premature translation termination and/ornonsense-mediated mRNA decay” refers to the regulation of geneexpression by altering the level of nonsense suppression. For example,if it is desirable to increase production of a defective protein encodedby a gene with a premature stop codon, i.e., to permit readthrough ofthe premature stop codon of the disease gene so translation of the genecan occur, then modulation of premature translation termination and/ornonsense-mediated mRNA decay entails up-regulation of nonsensesuppression. Conversely, if it is desirable to promote the degradationof an mRNA with a premature stop codon, then modulation of prematuretranslation termination and/or nonsense-mediated mRNA decay entailsdown-regulation of nonsense suppression.

As used herein, the term “patient” means an animal (e.g., cow, horse,sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guineapig, etc.), preferably a mammal such as a non-primate and a primate(e.g., monkey and human), most preferably a human. In certainembodiments, the patient is an infant, child, adolescent or adult. Inone embodiment, it has been determined through pre-screening that thepatient possesses a non-sense mutation. In another embodiment, it hasbeen determined through pre-screening which non-sense mutation thepatient has (i.e., UAA, UGA, or UAG). In another embodiment, the patientis infected with bacterial cells (e.g., Pseudomonas aeruginosa). Inanother embodiment, the cells of the patient are virally infected.

As used herein, unless otherwise specified, the term “substituted” meansa group substituted by one to four or more substituents, such as, halo,trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyoxy,heterocylooxy, oxo, alkanoyl, alkylcarbonyl, cycloalkyl, aryl, aryloxy,aralkyl, alkanoyloxy, cyano, azido, amino, alkylamino, arylamino,aralkylamino, cycloalkylamino, heterocycloamino, mono and disubstitutedamino in which the two substituents on the amino group are selected fromalkyl, aryl, aralkyl, alkanoylamino, aroylamino, aralkanoylamino,substituted alkanoylamino, substituted arylamino, substitutedaralkanoylamino, thiol, alkylthio, arylthio, aralkylthio,cycloalkylthio, heterocyclothio, alkylthiono, arylthiono, aralkylthiono,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, sulfonamido (e.g.,SO₂NH₂), substituted sulfonamido, nitro, carboxy, carbamyl (e.g. CONH₂),substituted carbamyl (e.g., CONH alkyl, CONH aryl, CONH aralkyl orinstances where there are two substituents on the nitrogen selected fromalkyl, aryl or aralkyl), alkoxycarbonyl, aryl, substituted aryl,guanidino and heterocyclo, such as, indolyl, imidazolyl, furyl, thienyl,thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Wherein, asnoted above, the substituents themselves are further substituted, suchfurther substituents are selected from the group consisting of halogen,alkyl, alkoxy, aryl and aralkyl. In a particular embodiment, the termsubstituted does not mean cyano.

As used herein, unless otherwise specified, the term “alkyl” means asaturated straight chain or branched non-cyclic hydrocarbon having from1 to 20 carbon atoms, preferably 1-10 carbon atoms and most preferably1-4 carbon atoms. Representative saturated straight chain alkyls include-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl,-n-octyl, -n-nonyl and -n-decyl; while saturated branched alkyls include-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl,2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl,5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl,2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl,2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl,3,3-dimtheylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl,3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl,2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl,2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl,2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyland the like. An alkyl group can be unsubstituted or substituted.Unsaturated alkyl groups include alkenyl groups and alkynyl groups,which are discussed below.

As used herein, unless otherwise specified the term “alkenyl group”means a straight chain or branched non-cyclic hydrocarbon having from 2to 20 carbon atoms, more preferably 2-10 carbon atoms, most preferably2-6 carbon atoms, and including at least one carbon-carbon double bond.Representative straight chain and branched (C₂-C₁₀)alkenyls include-vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl,-1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl,-3-octenyl, -1-nonenyl, -2-nonenyl, -3-nonenyl, -1-decenyl, -2-decenyl,-3-decenyl and the like. The double bond of an alkenyl group can beunconjugated or conjugated to another unsaturated group. An alkenylgroup can be unsubstituted or substituted.

As used herein, unless otherwise specified the term “alkynyl group”means a straight chain or branched non-cyclic hydrocarbon having from 2to 20 carbon atoms, more preferably 2-10 carbon atoms, most preferably2-6 carbon atoms, and including at least one carbon-carbon triple bond.Representative straight chain and branched —(C₂-C₁₀)alkynyls include-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl,-5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl,-2-octynyl, -7-octynyl, -1-nonynyl, -2-nonynyl, -8-nonynyl, -1-decynyl,-2-decynyl, -9-decynyl, and the like. The triple bond of an alkynylgroup can be unconjugated or conjugated to another unsaturated group. Analkynyl group can be unsubstituted or substituted.

As used herein, unless otherwise specified the term “halogen” or “halo”means fluorine, chlorine, bromine, or iodine.

As used herein, unless otherwise specified the term “alkyl sulfonyl”means -Alkyl-SO₃H or —SO₃-alkyl, wherein alkyl is defined as above,including —SO₂—CH₃, —SO₂—CH₂CH₃, —SO₂—(CH₂)₂CH₃, —SO₂—(CH₂)₃CH₃,—SO₂—(CH₂)₄CH₃, —SO₂—(CH₂)₅CH₃, and the like.

As used herein, unless otherwise specified the term “carboxyl” and“carboxy” mean —COOH.

As used herein, unless otherwise specified the term “alkoxy” means—O-(alkyl), wherein alkyl is defined above, including —OCH₃, —OCH₂CH₃,—O(CH₂)₂CH₃, —O (CH₂)₃CH₃, —O(CH₂)₄CH₃, —O(CH₂)₅CH₃, and the like.

As used herein, unless otherwise specified the term “alkoxycarbonyl”means —C(═O)O—(alkyl), wherein alkyl is defined above, including—C(═O)O—CH₃, —C(═O)O—CH₂CH₃, —C(═O)O—(CH₂)₂CH₃, —C(═O)O—(CH₂)₃CH₃,—C(═O)O—(CH₂)₄CH₃, —C(═O)O—(CH₂)₅CH₃, and the like. In a preferredembodiment, the esters are biohydrolyzable (i.e., the ester ishydrolyzed to a carboxylic acid in vitro or in vivo).

As used herein, unless otherwise specified the term “alkoxyalkyl” means-(alkyl)-O-(alkyl), wherein each “alkyl” is independently an alkyl groupas defined above, including —CH₂OCH₃, —CH₂OCH₂CH₃, —(CH₂)₂OCH₂CH₃,—(CH₂)₂O(CH₂)₂CH₃, and the like.

As used herein, unless otherwise specified the term “aryl” means acarbocyclic aromatic ring containing from 5 to 14 ring atoms. The ringatoms of a carbocyclic aryl group are all carbon atoms. Aryl ringstructures include compounds having one or more ring structures such asmono-, bi-, or tricyclic compounds as well as benzo-fused carbocyclicmoieties such as 5,6,7,8-tetrahydronaphthyl and the like. Preferably,the aryl group is a monocyclic ring or bicyclic ring. Representativearyl groups include phenyl, tolyl, anthracenyl, fluorenyl, indenyl,azulenyl, phenanthrenyl and naphthyl. A carbocyclic aryl group can beunsubstituted or substituted.

As used herein, unless otherwise specified the term “heteroaryl” means acarbocyclic aromatic ring containing from 5 to 14 ring atoms and thering atoms contain at least one heteroatom, preferably 1 to 3heteroatoms, independently selected from nitrogen, oxygen, or sulfur.Heteroaryl ring structures include compounds having one or more ringstructures such as mono-, bi-, or tricyclic compounds as well as fusedheterocycle moities. Representative heteroaryls are triazolyl,tetrazolyl, oxadiazolyl, pyridyl, furyl, benzofuranyl, thiophenyl,benzothiophenyl, benzoisoxazolyl, benzoisothiazolyl, quinolinyl,pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, benzoquinazolinyl, acridinyl, pyrimidyl andoxazolyl. A group can be unsubstituted or substituted.

As used herein, unless otherwise specified the term “aryloxy” means —O—aryl group, wherein aryl is as defined above. An aryloxy group can beunsubstituted or substituted.

As used herein, unless otherwise specified the term “arylalkyl” means-(alkyl)-(aryl), wherein alkyl and aryl are defined above, including,but not limited to —(CH₂)phenyl, —(CH₂)₂phenyl, —(CH₂)₃phenyl,—CH(phenyl)₂, —CH(phenyl)₃, —(CH₂)tolyl, —(CH₂)anthracenyl,—(CH₂)fluorenyl, —(CH₂)indenyl, —(CH₂)azulenyl, —(CH₂)naphthyl, and thelike.

As used herein, unless otherwise specified the term “heteroarylalkyl”means -(alkyl)-(heteroaryl), wherein alkyl and heteroaryl are definedabove, including, but not limited to —(CH₂)pyridyl, —(CH₂)₂pyridyl,—(CH₂)₃pyridyl, —CH(pyridyl)₂, —C(pyridyl)₃, —(CH₂)triazolyl,—(CH₂)tetrazolyl, —(CH₂)oxadiazolyl, —(CH₂)furyl, —(CH₂)benzofuranyl,—(CH₂)thiophenyl, —(CH₂)benzothiophenyl, and the like.

As used herein, unless otherwise specified the term “arylalkyloxy” means—O-(alkyl)-(aryl), wherein alkyl and aryl are defined above, including,but not limited to —O—(CH₂)₂-phenyl, —O—(CH₂)₃-phenyl, —O—CH(phenyl)₂,—O—CH(phenyl)₃, —O—(CH₂)tolyl, —O—(CH₂)anthracenyl, —O—(CH₂)fluorenyl,—O—(CH₂)indenyl, —O—(CH₂)azulenyl, —O—(CH₂)naphthyl, and the like.

As used herein, unless otherwise specified the term “cycloalkyl” means amonocyclic or polycyclic saturated ring comprising carbon and hydrogenatoms and having no carbon-carbon multiple bonds. A cycloalkyl group canbe unsubstituted or substituted. Examples of cycloalkyl groups include,but are not limited to, (C₃-C₇)cycloalkyl groups, including cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturatedcyclic and bicyclic terpenes. A cycloalkyl group can be unsubstituted orsubstituted. Preferably, the cycloalkyl group is a monocyclic ring orbicyclic ring.

As used herein, unless otherwise specified the term “heterocyclyl” meansa monocyclic or polycyclic ring comprising carbon and hydrogen atoms,optionally having 1 to 4 multiple bonds, and the ring atoms contain atleast one heteroatom, preferably 1 to 3 heteroatoms, independentlyselected from nitrogen, oxygen, and sulfur. Heterocyclyl ring structuresinclude compounds having one or more ring structures such as mono-, bi-,or tricylic compounds. Preferably, the heterocyclyl group is amonocyclic ring or bicyclic ring. Representative heterocycles include,but are not limited to morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like. A heterocyclyl ring can be unsubstituted or substituted.

As used herein, unless otherwise specified the term “cycloalkyloxy”means —O-(cycloalkyl), wherein cycloalkyl is defined above.

As used herein, unless otherwise specified the term “cycloalkylalkyloxy”means —O-(alkyl)-(cycloalkyl), wherein cycloalkyl and alkyl are definedabove, including, but not limited to —O-cyclopropyl, —O-cyclobutyl,—O-cyclopentyl, —O-cyclohexyl, —O-cycloheptyl and the like.

As used herein, unless otherwise specified the term “aminoalkoxy” means—O—(alkyl)-NH₂, wherein alkyl is defined above, including, but notlimited to —O—CH₂—NH₂, —O—(CH₂)₂—NH₂, —O—(CH₂)₃—NH₂, —O—(CH₂)₄—NH₂,—O—(CH₂)₅—NH₂, and the like.

As used herein, unless otherwise specified the term “alkylamino” means—NH(alkyl) or —N(alkyl)(alkyl), wherein alkyl is defined above,including, but not limited to NHCH₃, —NHCH₂CH₃, —NH(CH₂)₂CH₃,—NH(CH₂)₃CH₃, —NH(CH₂)₄—CH₃, —NH(CH₂)₅CH₃, —N(CH₃)₂, —N(CH₂CH₃)₂,—N((CH₂)₂CH₃)₂, —N(CH₃)(CH₂CH₃), and the like.

As used herein, unless otherwise specified the term “arylamino” means—NH(aryl), wherein aryl is defined above, including, but not limited to—NH(phenyl), —NH(tolyl), —NH(anthracenyl), —NH(fluorenyl), —NH(indenyl),—NH(azulenyl), —NH(pyridinyl), —NH(naphthyl), and the like.

As used herein, unless otherwise specified the term “arylalkylamino”means —NH-(alkyl)-(aryl), wherein alkyl and aryl are defined above,including —NH—CH₂-(phenyl), —NH—CH₂-(tolyl), —NH—CH₂-(anthracenyl),—NH—CH₂-(fluorenyl), —NH—CH₂-(indenyl), —NH—CH₂-(azulenyl),—NH—CH₂-(pyridinyl), —NH—CH₂-naphthyl), —NH—(CH₂)₂-(phenyl) and thelike.

As used herein, unless otherwise specified the term “cycloalkylamino”means —NH-(cycloalkyl), wherein cycloalkyl is defined above, including—NH-cyclopropyl, —NH-cyclobutyl, —NH-cyclopentyl, —NH-cyclohexyl,—NH-cycloheptyl, and the like.

As used herein, unless otherwise specified the term “aminoalkyl” means-(alkyl)-NH₂, wherein alkyl is defined above, including —CH₂—NH₂,—(CH₂)₂—NH₂, —(CH₂)₃—NH₂, —(CH₂)₄—NH₂, —(CH₂)₅—NH₂ and the like.

As used herein, unless otherwise specified the term “alkylaminoalkyl”means -(alkyl)-NH(alkyl) or -(alkyl)-N(alkyl)(alkyl), wherein each“alkyl” is independently an alkyl group defined above, including—CH₂—NH—CH₃, —CH₂—NHCH₂CH₃, —CH₂—NH(CH₂)₂CH₃, —CH₂—NH(CH₂)₃CH₃,—CH₂—NH(CH₂)₄—CH₃, —CH₂—NH(CH₂)₅CH₃, —(CH₂)₂—NH—CH₃, —CH₂—N(CH₃)₂,—CH₂—N(CH₂CH₃)₂, —CH₂—N((CH₂)₂CH₃)₂, —CH₂—N(CH₃)(CH₂CH₃),—(CH₂)₂—N(CH₃)₂, and the like.

As used herein, a “therapeutically effective amount” refers to thatamount of the compound of the invention or other active ingredientsufficient to provide a therapeutic benefit in the treatment ormanagement of the disease or to delay or minimize symptoms associatedwith the disease. Further, a therapeutically effective amount withrespect to a compound of the invention means that amount of therapeuticagent alone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of the disease. Usedin connection with an amount of a compound of the invention, the termcan encompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of disease, or enhances the therapeutic efficacy ofor synergies with another therapeutic agent.

As used herein, a “prophylactically effective amount” refers to thatamount of a compound of the invention or other active ingredientsufficient to result in the prevention, recurrence or spread of thedisease. A prophylactically effective amount may refer to the amountsufficient to prevent initial disease or the recurrence or spread of thedisease or the occurrence of the disease in a patient, including but notlimited to those predisposed to the disease. A prophylacticallyeffective amount may also refer to the amount that provides aprophylactic benefit in the prevention of the disease. Further, aprophylactically effective amount with respect to a compound of theinvention means that amount alone, or in combination with other agents,that provides a prophylactic benefit in the prevention of the disease.Used in connection with an amount of a compound of the invention, theterm can encompass an amount that improves overall prophylaxis orenhances the prophylactic efficacy of or synergies with anotherprophylactic agent.

As used herein, a “therapeutic protocol” refers to a regimen of timingand dosing of one or more therapeutic agents.

As used herein, a “prophylactic protocol” refers to a regimen of timingand dosing of one or more prophylactic agents.

A used herein, a “protocol” includes dosing schedules and dosingregimens.

As used herein, “in combination” refers to the use of more than oneprophylactic and/or therapeutic agents.

As used herein, the terms “manage”, “managing” and “management” refer tothe beneficial effects that a subject derives from a prophylactic ortherapeutic agent, which does not result in a cure of the disease. Incertain embodiments, a subject is administered one or more prophylacticor therapeutic agents to “manage” a disease so as to prevent theprogression or worsening of the disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the onset recurrence, spread or of the disease in asubject resulting from the administration of a prophylactic ortherapeutic agent.

As used herein, the terms “treat”, “treating” and “treatment” refer tothe eradication or amelioration of the disease or symptoms associatedwith the disease. In certain embodiments, such terms refer to minimizingthe spread or worsening of the disease resulting from the administrationof one or more prophylactic or therapeutic agents to a subject with sucha disease.

As used herein, the term “pharmaceutically acceptable salts” refer tosalts prepared from pharmaceutically acceptable non-toxic acids or basesincluding inorganic acids and bases and organic acids and bases.Suitable pharmaceutically acceptable base addition salts for thecompound of the present invention include, but are not limited to,metallic salts made from aluminum, calcium, lithium, magnesium,potassium, sodium and zinc or organic salts made from lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitablenon-toxic acids include, but are not limited to, inorganic and organicacids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic,galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic,phosphoric, propionic, salicylic, stearic, succinic, sulfanilic,sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific non-toxicacids include hydrochloric, hydrobromic, phosphoric, sulfuric, andmethanesulfonic acids. Examples of specific salts thus includehydrochloride and mesylate salts. Other examples of salts are well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, 18th ed.,Mack Publishing, Easton Pa. (1990).

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide anactive compound, particularly a compound of the invention. Examples ofprodrugs include, but are not limited to, derivatives and metabolites ofa compound of the invention that include biohydrolyzable moieties suchas biohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, biohydrolyzable ureides, andbiohydrolyzable phosphate analogues. Preferably, prodrugs of compoundswith carboxyl functional groups are the lower alkyl esters of thecarboxylic acid. The carboxylate esters are conveniently formed byesterifying any of the carboxylic acid moieties present on the molecule.Prodrugs can typically be prepared using well-known methods, such asthose described by Burger's Medicinal Chemistry and Drug Discovery 6thed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application ofProdrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters,and choline esters. Examples of biohydrolyzable amides include, but arenot limited to, lower alkyl amides, α-amino acid amides, alkoxyacylamides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzablecarbamates include, but are not limited to, lower alkylamines,substituted ethylenediamines, aminoacids, hydroxyalkylamines,heterocycle and heteroaromatic amines, and polyether amines.

As used herein and unless otherwise indicated, the term “optically pure”or “stereomerically pure” means a the stereoisomer of a compound issubstantially free of the other stereoisomers of that compound. Forexample, a stereomerically pure compound having one chiral center willbe substantially free of the opposite enantiomer of the compound. Astereomerically pure compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, more preferably greater than about90% by weight of one stereoisomer of the compound and less than about10% by weight of the other stereoisomers of the compound, even morepreferably greater than about 95% by weight of one stereoisomer of thecompound and less than about 5% by weight of the other stereoisomers ofthe compound, and most preferably greater than about 97% by weight ofone stereoisomer of the compound and less than about 3% by weight of theother stereoisomers of the compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure” means a stereomerically pure composition of acompound having one chiral center.

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Compounds of the Invention

This invention encompasses 1,2,4-oxadiazole benzoic acid compounds offormula I:

or pharmaceutically acceptable salts, hydrates, clathrates, prodrugs,polymorphs, stereoisomers, including enantiomers, diastereomers,racemates or mixtures of stereoisomers, thereof wherein:

Z is substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted heterocycle, substituted or unsubstituted arylalkyl;

R¹ is hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, —(CH₂CH₂O)_(n)R⁶ or any biohydrolyzable group;

R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl; substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy,aryloxy, heteroaryloxy, halogen, CF₃, OCF₃, OCHF₂, CN, COOH, COOR⁷,SO₂R⁷, NO₂, NH₂, or N(R⁷)₂;

each occurrence of R⁷ is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl; substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy,aryloxy, heteroaryloxy, halogen or CF₃; and

n is an integer from 1 to 7.

In an alternative embodiment, the invention encompasses a compound ofFormula I wherein when R¹, R², R³, R⁴, and R⁵ are hydrogen, Z is notmethyl, 2-carboxy ethyl, 3-(4-pyridinyl)propyl, or2-(4-piperidinyl)ethyl.

In a preferred embodiment, the invention encompasses a compound ofFormula I wherein R¹ is H.

In a preferred embodiment; the invention encompasses a compound ofFormula I wherein R¹ is any biohydrolyzable group other than H.

In a related embodiment, the invention encompasses 1,2,4-oxadiazolebenzoic acid compounds of the formula II:

or pharmaceutically acceptable salts, hydrates, clathrates, orstereoisomers thereof Z is substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted heterocycle,substituted or unsubstituted arylalkyl; and R is hydrogen or halogen.

In one embodiment R is the halogen, fluorine. In a preferred embodiment,R is hydrogen.

In a preferred embodiment, the invention encompasses a compound ofFormula I or II wherein Z is p-Tolyl; (4-Chloromethyl-phenyl);(2-Chloro-pyridin-3-yl); (2-Fluoro-phenyl); (3,4-Difluoro-phenyl);(4-Methoxy-phenyl); Benzo[1,3]dioxol-yl; (4-Ethyl-phenyl); o-Tolyl;(2-Chloro-phenyl); (3-Methyl-thiophen-2-yl); Benzo[b]thiophen-2-yl;(3-Fluoro-phenyl); (4-tert-Butyl-phenyl); (2-Methoxy-phenyl(2,5-Difluoro-phenyl); Thiophen-2-yl; (2,4-Difluoro-phenyl);(3-Chloro-phenyl); m-Tolyl; (4-Trifluoromethyl-phenyl);(4-Fluoro-phenyl); (3-Methoxy-phenyl); Phenyl; (2,6-Difluoro-phenyl);(2,5-Dimethyl-furan-3-yl); (4-Pyrrol-1-yl-phenyl);(3-Dimethylamino-phenyl); Biphenyl-4-yl; (4-Dimethylamino-phenyl);Benzo[1,2,5]oxadiazol-yl; m-Tolyl; (2-Trifluoromethyl-phenyl);(6-Chloro-pyridin-3-yl); (3,5-Bis-trifluoromethyl-phenyl); Furan-2-yl;(4-Nitro-phenyl); (3,4-Dimethoxy-phenyl); (3-Trifluoromethoxy-phenyl);Naphthalen-1-yl; Cyclohexyl; Pyridin-3-yl; Pyridin-4-yl; Cyclopentyl;Cyclopropyl; (4-Pentyloxy-phenyl); (3,4,5-Trimethoxy-phenyl);(4-Isobutyl-phenyl); Cyclobutyl; 5-(1-Acetyl-piperidin-4-yl);5-Isoxazol-5-yl; [3-(2-Chloro-6-fluoro-phenyl)-5-methyl-isoxazol-4-yl]or [3-(2-Chloro-phenyl)-5-methyl-isoxazol-4-yl]; more preferably Z is(3-Fluoro-phenyl), more preferably Z is (4-Fluoro-phenyl), even morepreferably Z is (2-Fluoro-phenyl).

In a specific embodiment, the invention encompasses a compound ofFormula I or II wherein Z is not 4-cyano-phenyl.

Preferred compounds of the invention include, but are not limited to,

3-(5-p-Tolyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Chloromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2-Chloro-pyridin-3-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3,4-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Benzo[1,3]dioxol-5-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Ethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-o-Tolyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(2-Chloro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Methyl-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Benzo[b]thiophen-2-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(3-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-tert-Butyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2,5-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Thiophen-2-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(2,4-Difluoro-phenyl)-[1,2,4]-oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Chloro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-m-Tolyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Phenyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(2,6-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2,5-Dimethyl-furan-3-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Pyrrol-1-yl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Dimethylamino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Biphenyl-4-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Dimethylamino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Benzo[1,2,5]oxadiazol-5-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-m-Tolyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(2-Trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(6-Chloro-pyridin-3-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3,5-Bis-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid;

3-(5-Furan-2-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Nitro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3,4-Dimethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Naphthalen-1-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-Cyclohexyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-Pyridin-3-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-Pyridin-4-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-Cyclopentyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-Cyclopropyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Pentyloxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3,4,5-Trimethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Isobutyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Cyclobutyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(1-Acetyl-piperidin-4-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Isoxazol-5-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-{5-[3-(2-Chloro-6-fluoro-phenyl)-5-methyl-isoxazol-4-yl]-[1,2,4]oxadiazol-3-yl}-benzoicacid;

3-(5-Isopropyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-tert-Butyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Butyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-Propenyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Chloro-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Chloro-phenoxymethyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Benzyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-(5-Methoxymethyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(1-Phenyl-propyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Fluoro-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Chloro-phenoxymethyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(6-Chloro-pyridin-3-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-(5-Cyclopentylmethyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;

3-[5-(4-Methoxy-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2,3-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2-Fluoro-5-methyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2-Methylsulfanyl-pyridin-3-yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid;

3-[5-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid;

4-Fluoro-3-[5-(4-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

2-Fluoro-5-[5-(4-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Chloro-2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Bromo-2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Fluoro-biphenyl-4-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-{5-[3-(2-Chloro-phenyl)-5-methyl-isoxazol-4-yl]-[1,2,4]oxadiazol-3-yl}-benzoicacid;

3-[5-(4-Cyano-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid sodium salt;

3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester;

5-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-2-methoxy-benzoic acid;

3-[5-(4-Bromo-2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(3-Fluoro-biphenyl-4-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(6-Pyrrolidin-1-yl-pyridin-3-yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid;

3-[5-(6-Morpholin-4-yl-pyridin-3-yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid;

3-[5-(3,4,5,6-Tetrahydro-2H-[1,2′]bipyridinyl-5′-yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid;

3-[5-(2-Fluoro-6-hydroxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-methoxy-ethyl ester;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-methoxy-ethoxy)-ethyl ester;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethyl ester;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl ester;

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-[2-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethylester;

3-[5-(4-Amino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;

3-[5-(4-Azido-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid; and

3-[5-(4-Benzyloxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid andpharmaceutically acceptable salts, hydrates, solvates, clathrates andstereoisomers thereof.

The compounds of formulas I and II and those listed above are hereinreferred to as “compounds of the invention”. Exemplary compounds of theinvention are depicted in Table 1 below.

TABLE I Compound Compound Name Activity 01

3-[5-(3-Chloro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 02

3-[5-(4-Pentyloxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid *** 03

3-(5-Naphthalen-1-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid **** 04

3-(5-p-Tolyl-[1,2,4]oxadiazol-3- yl)-benzoic acid ***** 05

3-[5-(4-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 06

3-(5-Biphenyl-4-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid **** 07

3-[5-(4-isobutyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ** 08

3-(5-Phenyl-[1,2,4]oxadiazol-3- yl)-benzoic acid ***** 09

3-(5-Cyclohexyl-[1,2,4]oxadiazol- 3-yl)-benzoic acid **** 10

3-[5-(3,4,5-Trimethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***11

3-[5-(4-Nitro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 12

3-[5-(4-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 13

3-[5-(o-tolyl)-[1,2,4]oxadiazol-3- yl]-benzoic acid ***** 14

3-(5-Benzo[1,3]dioxol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid ***** 15

3-(5-Isopropyl-[1,2,4]oxadiazol-3- yl)-benzoic acid **** 16

3-[5-(3,5-Bis-trifluoromethyl- phenyl)-[1,2,4]oxadiazol-3-yl]- benzoicacid **** 17

3-[5-(4-Trifluoromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid***** 18

3-[5-(4-Dimethylamino-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ****19

3-[5-(2-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid *** 20

3-[5-(3-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 21

3-(5-Furan-2-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid **** 22

3-(5-tert-Butyl-[1,2,4]oxadiazol-3- yl)-benzoic acid * 23

3-(5-Benzo[1,2,4]oxadiazol-5-yl- [1,2,5]oxadiazol-3-yl)-benzoic acid**** 24

3-[5-(4-Chloromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid *****25

3-[5-(4-tert-Butyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 26

3-(5-Butyl-[1,2,4]oxadiazol-3-yl)- benzoic acid **** 27

3-(5-Cyclopropyl- [1,2,4]oxadiazol-3-yl)-benzoic acid **** 28

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 29

3-(5-Thiophen-2-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid **** 30

3-(5-Propenyl-[1,2,4]oxadiazol-3- yl)-benzoic acid **** 31

3-(5-Cyclopentyl- [1,2,4]oxadiazol-3-yl)-benzoic acid **** 32

3-(5-Thiophen-2-ylmethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid **** 33

3-[5-(4-Chloro-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 34

3-[5-(4-Chloro-phenoxymethyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ****35

3-[5-(2-Trifluoromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid**** 36

3-[5-(2,6-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 37

3-[5-(3-Fluoro-phenyl)- [1,2,4]oxaediazol-3-yl]-benzoic acid ***** 38

3-[5-(4-Ethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 39

3-[5-(3,4-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 40

3-(5-m-Tolyl-[1,2,4]oxadiazol-3- yl)-benzoic acid ***** 41

3-[5-(4-Pyrrol-1-yl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 42

3-(5-Benzyl-[1,2,4]oxadiazol-3- yl)-benzoic acid *** 43

3-(5-Methoxymethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid * 44

3-[5-(2,5-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 45

3-[5-(1-Phenyl-propyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid * 46

3-[5-(2-Chloro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 47

3-[5-(3-Trifluoromethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid**** 48

3-[5-(4-Fluoro-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 49

3-[5-(2,5-Dimethyl-furan-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ****50

3-[5-(3-Methyl-thiophen-2-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid *****51

3-[5-(3-Chloro-phenoxymethyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ** 52

3-(5-Isoxazol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid ** 53

3-[5-(6-Chloro-pyridin-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ****54

3-{5-[3-(2-Chloro-phenyl)-5- methyl-isoxazol-4-yl]-[1,2,4]oxadiazol-3-yl}-benzoic acid 55

3-{5-[3-(2-Chloro-6-fluoro- phenyl)-5-methyl-isoxazol-4-yl]-[1,2,4]oxadiazol-3-yl}-benzoic acid 56

3-(5-Cyclopentylmethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid *** 57

3-[5-(2,4-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 58

3-(5-Pyridin-3-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid *** 59

3-(5-Pyridin-4-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid *** 60

3-(5-Cyclobutyl-[1,2,4]oxadiazol- 3-yl)-benzoic acid ** 61

3-[5-(4-Methoxy-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ** 62

3-[5-(3,4-Dimethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 63

3-[5-(2-Chloro-pyridin-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ****64

3-[5-(1-Acetyl-piperidin-4-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid * 65

3-(5-Benzo[b]thiophen-2-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid ***** 66

3-[5-(3-Dimethylamino-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ****67

3-[5-(2,3-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 68

3-[5-(2-Fluoro-5-methyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***69

3-[5-(2-Methylsulfanyl-pyridin-3- yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid **** 70

3-[5-(2,2-Difluoro- benzo[1,3]dioxol-5-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid *** 71

4-Fluoro-3-[5-(4-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid *72

2-Fluoro-5-[5-(4-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***73

3-[5-(4-Chloro-2-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid**** 74

3-[5-(4-Bromo-2-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***75

3-[5-(3-Fluoro-biphenyl-4-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ****76

5-[5-(4-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-2- methoxy-benzoic acid *77

3-[5-(4-Cyano-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 78

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid sodium salt***** 79

3-[5-(4-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester*** 80

5-[5-(4-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-2-methoxy- benzoic acid *81

3-[5-(3-Fluoro-biphenyl-4-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ** 82

3-[5-(6-Pyrrolidin-1-yl-pyridin-3- yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid **** 83

3-[5-(6-Morpholin-4-yl-pyridin-3- yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid **** 84

3-[5-(3,4,5,6-Tetrahydro-2H- [1,2′]bipyridinyl-5′-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid **** 85

3-[5-(2-Fluoro-6-hydroxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **86

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester** 87

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid2-methoxy-ethyl ester *** 88

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-methoxy-ethoxy)-ethyl ester ** 89

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxaediazol-3-yl]-benzoic acid2-[2-(2-methoxy-ethoxy)- ethoxy]-ethyl ester ** 90

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid2-{2-[2-(2-methoxy-ethoxy)- ethoxy]-ethoxy}-ethyl ester ** 91

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-{2-[2-(2-methoxy- ethoxy)-ethoxy]-ethoxy}-ethoxy)- ethyl ester ***92

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid2-[2-(2-{2-[2-(2-hydroxy- ethoxy)-ethoxy]-ethoxy}-ethoxy)- ethoxy]-ethylester *** 93

3-[5-(4-Amino-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid **** 94

3-[5-(4-Azido-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid ***** 95

3-[5-(4-Benzyloxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid *

Activity measurements in Table I were performed in a cell-basedluciferase reporter assay (as described in Section 4.2) comprising aluciferase reporter construct containing a UGA premature terminationcodon that was stably transfected in 293T Human Embryonic Kidney cells.A small molecule,3-[3-(4-Isopropyl-phenyl)-2,5-dioxo-imidazolidin-1-yl]-benzoic acid,known to allow readthrough of premature termination codons was used wasused as an internal standard. Activity measurements are based on thequalitative relation between the minimum concentration of compoundrequired to produce a given protein in a cell (potency) and the maximumamount of protein produced by the cell (efficacy). Potency and efficacyactivities are ranked as either extremely high, very high orsignificant. The combination of these activities is used to determinethe activity ranking. Compounds which were found to have both extremelyhigh potency and extremely high efficacy of protein synthesis areclassified as “*****”. Compounds which were found to have extremely highpotency of protein synthesis and very high efficacy were classified as“****”. Compounds which were found to have very high potency of proteinsynthesis and extremely high efficacy were classified as “****”.Compounds which were found to have both very high potency and very highefficacy of protein synthesis are classified as “***”. Compounds whichwere found to have very high potency of protein synthesis andsignificant efficacy were classified as “**”. Compounds which were foundto have significant potency of protein synthesis and very high efficacywere classified as “**”. Similarly, compounds which were found to havesignificant potency and efficacy of protein synthesis were classified as“*” (see table below).

Potency Efficacy Ranking Extremely high Extremely high ***** Extremelyhigh Very high **** Very high Extremely high **** Very high Very high*** Very high Significant ** Significant Very high ** SignificantSignificant *

Compounds having less than significant potency or efficacy of proteinsynthesis or both in the cell-based luciferase assay were classifiedwith no asterisks. Nevertheless, these compounds are believed to haveutility in the in vivo methods of the invention.

The present invention encompasses the in vitro or in vivo use of acompound of the invention, and the incorporation of a compound of theinvention into pharmaceutical compositions and single unit dosage formsuseful in the treatment and prevention of a variety of diseases anddisorders. Specific diseases and disorders include those ameliorated bythe suppression of a nonsense mutation in messenger RNA.

Pharmaceutical compositions including dosage forms of the invention,which comprise a compound of the invention or a pharmaceuticallyacceptable polymorph, prodrug, salt, clathrate, solvate or hydratethereof, can be used in the methods of the invention.

Without being limited by theory, it is believed that a compound of theinvention can modulate premature translation termination and/ornonsense-mediated mRNA decay. Consequently, a first embodiment of theinvention relates to a method of modulating premature translationtermination and/or nonsense-mediated mRNA decay comprising contacting acell exhibiting a nonsense mutation with an effective amount of acompound of the invention, or a pharmaceutically acceptable prodrug,metabolite, polymorph, salt, solvate, hydrate, or clathrate thereof. Ina particular embodiment, the invention relates to a method of inducingnonsense suppression comprising contacting a cell exhibiting a nonsensemutation with an effective amount of a compound of the invention, or apharmaceutically acceptable prodrug, metabolite, polymorph, salt,solvate, hydrate, or clathrate thereof.

4.2 Biological Assays and Animal Studies

Compounds that modulate premature translation termination and/ornonsense-mediated mRNA decay can be identified by a number oftechniques. For example, methods for screening compounds that modulatethe post-transcriptional expression of any gene with a prematuretranslation stop codon are described in International Patent PublicationNo. WO 01/44516 A2, incorporated herein in its entirety by reference. Ina preferred embodiment, a mRNA with a premature termination codon istranslated in vitro and is used to screen a library of test compounds.In a preferred embodiment, the mRNA with a premature termination codonis a reporter gene with a premature termination codon.

Two assays were developed for use in high throughput screens to identifysmall molecules that promote nonsense suppression. Each assay utilizedluciferase because it is a functional reporter gene assay (light is onlyproduced if the protein is functional) and it is extremely sensitive(Light intensity is proportional to luciferase concentration in the nMrange). The first assay is a cell-based luciferase reporter assay andthe second is a biochemical assay consisting of rabbit reticulocytelysate and a nonsense-containing luciferase reporter mRNA. In thecell-based assay, a luciferase reporter construct containing a UGApremature termination codon was stably transfected in 293T HumanEmbryonic Kidney cells. In the biochemical assay, mRNA containing a UGApremature termination codon was used as a reporter in an in vitrotranslation reaction using rabbit reticulocyte lysate supplemented withtRNA, hemin, creatine kinase, amino acids, KOAc, Mg(OAc)₂, and creatinephosphate. Translation of the mRNA was initiated within a virus derivedleader sequence, which significantly reduced the cost of the assaybecause capped RNA was not required. Synthetic mRNA was prepared invitro using the T7 promoter and the MegaScript in vitro transcriptionkit (Ambion). In both of the biochemical and cell-based assays, additionof a small molecule known to allow readthrough of premature terminationcodons, 3-[3-(4-Isopropyl-phenyl)-2,5-dioxo-imidazolidin-1-yl]-benzoicacid, resulted in increased luciferase activity and was, therefore, usedas an internal standard.

Animal model systems can also be used to demonstrate the safety andefficacy of compounds of formula I or II. The compounds of formula I orII can be tested for biological activity using animal models for adisease, condition, or syndrome of interest. These include animalsengineered to contain the target RNA element coupled to a functionalreadout system, such as a transgenic mouse.

Examples of animal models for cystic fibrosis include, but are notlimited to, cftr(−/−) mice (see, e.g., Freedman et al., 2001,Gastroenterology 121(4):950-7), cftr(tm1HGU/tm1HGU) mice (see, e.g.,Bernhard et al., 2001, Exp Lung Res 27(4):349-66), CFTR-deficient micewith defective cAMP-mediated Cl(−) conductance (see, e.g., Stotland etal., 2000, Pediatr Pulmonol 30(5):413-24), andC57BL/6-Cftr(mIUNC)/Cftr(m1UNC) knockout mice (see, e.g., Stotland etal., 2000, Pediatr Pulmonol 30(5):413-24).

Examples of animal models for muscular dystrophy include, but are notlimited to, mouse, hamster, cat, dog, and C. elegans. Examples of mousemodels for muscular dystrophy include, but are not limited to, the dy−/−mouse (see, e.g., Connolly et al., 2002, J Neuroimmunol 127(1-2):80-7),a muscular dystrophy with myositis (mdm) mouse mutation (see, e.g.,Garvey et al., 2002, Genomics 79(2):146-9), the mdx mouse (see, e.g.,Nakamura et al., 2001, Neuromuscul Disord 11(3):251-9), theutrophin-dystrophin knockout (dko) mouse (see, e.g., Nakamura et al.,2001, Neuromuscul Disord 11(3):251-9), the dy/dy mouse (see, e.g.,Dubowitz et al., 2000, Neuromuscul Disord 10(4-5):292-8), the mdx(Cv3)mouse model (see, e.g., Pillers et al., 1999, Laryngoscope109(8):1310-2), and the myotonic ADR-MDX mutant mice (see, e.g., Krameret al., 1998, Neuromuscul Disord 8(8):542-50). Examples of hamstermodels for muscular dystrophy include, but are not limited to,sarcoglycan-deficient hamsters (see, e.g., Nakamura et al., 2001, Am JPhysiol Cell Physiol 281(2):C690-9) and the BIO 14.6 dystrophic hamster(see, e.g., Schlenker & Burbach, 1991, J Appl Physiol 71(5):1655-62). Anexample of a feline model for muscular dystrophy includes, but is notlimited to, the hypertrophic feline muscular dystrophy model (see, e.g.,Gaschen & Burgunder, 2001, Acta Neuropathol (Berl) 101(6):591-600).Canine models for muscular dystrophy include, but are not limited to,golden retriever muscular dystrophy (see, e.g., Fletcher et al., 2001,Neuromuscul Disord 11(3):239-43) and canine X-linked muscular dystrophy(see, e.g., Valentine et al., 1992, Am J Med Genet. 42(3):352-6).Examples of C. elegans models for muscular dystrophy are described inChamberlain & Benian, 2000, Curr Biol 10(21):R795-7 and Culette &Sattelle, 2000, Hum Mol Genet. 9(6):869-77.

Examples of animal models for familial hypercholesterolemia include, butare not limited to, mice lacking functional LDL receptor genes (see,e.g., Aji et al., 1997, Circulation 95(2):430-7), Yoshida rats (see,e.g., Fantappie et al., 1992, Life Sci 50(24):1913-24), the JCR:LA-cprat (see, e.g., Richardson et al., 1998, Atherosclerosis 138(1):135-46),swine (see, e.g., Hasler-Rapacz et al., 1998, Am J Med Genet.76(5):379-86), and the Watanabe heritable hyperlipidaemic rabbit (see,e.g., Tsutsumi et al., 2000, Arzneimittelforschung 50(2):118-21; Harschet al., 1998, Br J Pharmacol 124(2):227-82; and Tanaka et al., 1995,Atherosclerosis 114(1):73-82).

An example of an animal model for human cancer in general includes, butis not limited to, spontaneously occurring tumors of companion animals(see, e.g., Vail & MacEwen, 2000, Cancer Invest 18(8):781-92). Examplesof animal models for lung cancer include, but are not limited to, lungcancer animal models described by Zhang & Roth (1994, In Vivo8(5):755-69) and a transgenic mouse model with disrupted p53 function(see, e.g., Morris et al., 1998, J La State Med Soc 150(4):179-85). Anexample of an animal model for breast cancer includes, but is notlimited to, a transgenic mouse that overexpresses cyclin D1 (see, e.g.,Hosokawa et al., 2001, Transgenic Res 10(5):471-8). An example of ananimal model for colon cancer includes, but is not limited to, a TCRbetaand p53 double knockout mouse (see, e.g., Kado et al., 2001, Cancer Res61(6):2395-8). Examples of animal models for pancreatic cancer include,but are not limited to, a metastatic model of Panc02 murine pancreaticadenocarcinoma (see, e.g., Wang et al., 2001, Int J Pancreatol29(1):37-46) and nu-nu mice generated in subcutaneous pancreatic tumours(see, e.g., Ghaneh et al., 2001, Gene Ther 8(3):199-208). Examples ofanimal models for non-Hodgkin's lymphoma include, but are not limitedto, a severe combined immunodeficiency (“SCID”) mouse (see, e.g., Bryantet al., 2000, Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenicmouse (see, e.g., Hough et al., 1998, Proc Natl Acad Sci USA95(23):13853-8). An example of an animal model for esophageal cancerincludes, but is not limited to, a mouse transgenic for the humanpapillomavirus type 16 E7 oncogene (see, e.g., Herber et al., 1996, JVirol 70(3):1873-81). Examples of animal models for colorectalcarcinomas include, but are not limited to, Apc mouse models (see, e.g.,Fodde & Smits, 2001, Trends Mol Med 7(8):369-73 and Kuraguchi et al.,2000, Oncogene 19(50):5755-63). An example of an animal model forneurofibromatosis includes, but is not limited to, mutant NF1 mice (see,e.g., Cichowski et al., 1996, Semin Cancer Biol 7(5):291-8). Examples ofanimal models for retinoblastoma include, but are not limited to,transgenic mice that expression the simian virus 40 T antigen in theretina (see, e.g., Howes et al., 1994, Invest Opthalmol V is Sci35(2):342-51 and Windle et al, 1990, Nature 343(6259):665-9) and inbredrats (see, e.g., Nishida et al., 1981, Curr Eye Res 1(1):53-5 andKobayashi et al., 1982, Acta Neuropathol (Berl) 57(2-3):203-8). Examplesof animal models for Wilm's tumor include, but are not limited to, a WT1knockout mice (see, e.g., Scharnhorst et al., 1997, Cell Growth Differ8(2):133-43), a rat subline with a high incidence of neuphroblastoma(see, e.g., Mesfin & Breech, 1996, Lab Anim Sci 46(3):321-6), and aWistar/Furth rat with Wilms' tumor (see, e.g., Murphy et al., 1987,Anticancer Res 7(4B):717-9).

Examples of animal models for retinitis pigmentosa include, but are notlimited to, the Royal College of Surgeons (“RCS”) rat (see, e.g.,Vollrath et al., 2001, Proc Natl Acad Sci USA 98(22); 12584-9 andHanitzsch et al., 1998, Acta Anat (Basel) 162(2-3):119-26), a rhodopsinknockout mouse (see, e.g., Jaissle et al., 2001, Invest Opthalmol VisSci 42(2):506-13), and Wag/Rij rats (see, e.g., Lai et al., 1980, Am JPathol 98(1):281-4).

Examples of animal models for cirrhosis include, but are not limited to,CCl₄-exposed rats (see, e.g., Kloehn et al., 2001, Horm Metab Res33(7):394-401) and rodent models instigated by bacterial cell componentsor colitis (see, e.g., Vierling, 2001, Best Pract Res Clin Gastroenterol15(4):591-610).

Examples of animal models for hemophilia include, but are not limitedto, rodent models for hemophilia A (see, e.g., Reipert et al., 2000,Thromb Haemost 84(5):826-32; Jarvis et al., 1996, Thromb Haemost75(2):318-25; and Bi et al., 1995, Nat Genet. 10(1):119-21), caninemodels for hemophilia A (see, e.g., Gallo-Penn et al., 1999, Hum GeneTher 10(11):1791-802 and Connelly et al, 1998, Blood 91(9); 3273-81),murine models for hemophilia B (see, e.g., Snyder et al., 1999, Nat Med5(1):64-70; Wang et al., 1997, Proc Natl Acad Sci USA 94(21):11563-6;and Fang et al., 1996, Gene Ther 3(3):217-22), canine models forhemophilia B (see, e.g., Mount et al., 2002, Blood 99(8):2670-6; Snyderet al., 1999, Nat Med 5(1):64-70; Fang et al., 1996, Gene Ther3(3):217-22); and Kay et al., 1994, Proc Natl Acad Sci USA91(6):2353-7), and a rhesus macaque model for hemophilia B (see, e.g.,Lozier et al., 1999, Blood 93(6):1875-81).

Examples of animal models for von Willebrand disease include, but arenot limited to, an inbred mouse strain RIIIS/J (see, e.g., Nichols etal., 1994, 83(11):3225-31 and Sweeney et al., 1990, 76(11):2258-65),rats injected with botrocetin (see, e.g., Sanders et al., 1988, LabInvest 59(4):443-52), and porcine models for von Willebrand disease(see, e.g., Nichols et al., 1995, Proc Natl Acad Sci USA 92(7):2455-9;Johnson & Bowie, 1992, J Lab Clin Med 120(4):553-8); and Brinkhous etal., 1991, Mayo Clin Proc 66(7):733-42).

Examples of animal models for b-thalassemia include, but are not limitedto, murine models with mutations in globin genes (see, e.g., Lewis etal., 1998, Blood 91(6):2152-6; Raja et al., 1994, Br J Haematol86(1):156-62; Popp et al., 1985, 445:432-44; and Skow et al., 1983, Cell34(3):1043-52).

Examples of animal models for kidney stones include, but are not limitedto, genetic hypercalciuric rats (see, e.g., Bushinsky et al., 1999,Kidney Int 55(1):234-43 and Bushinsky et al., 1995, Kidney Int48(6):1705-13), chemically treated rats (see, e.g., Grases et al., 1998,Scand J Urol Nephrol 32(4):261-5; Burgess et al., 1995, Urol Res23(4):239-42; Kumar et al., 1991, J Urol 146(5):1384-9; Okada et al.,1985, Hinyokika Kiyo 31(4):565-77; and Bluestone et al., 1975, LabInvest 33(3):273-9), hyperoxaluric rats (see, e.g., Jones et al., 1991,J Urol 145(4):868-74), pigs with unilateral retrograde flexiblenephroscopy (see, e.g., Seifmah et al., 2001, 57(4):832-6), and rabbitswith an obstructed upper urinary tract (see, e.g., Itatani et al., 1979,Invest Urol 17(3):234-40).

Examples of animal models for ataxia-telangiectasia include, but are notlimited to, murine models of ataxia-telangiectasia (see, e.g., Barlow etal., 1999, Proc Natl Acad Sci USA 96(17):9915-9 and Inoue et al., 1986,Cancer Res 46(8):3979-82).

Examples of animal models for lysosomal storage diseases include, butare not limited to, mouse models for mucopolysaccharidosis type VII(see, e.g., Brooks et al., 2002, Proc Natl Acad Sci USA. 99(9):6216-21;Monroy et al., 2002, Bone 30(2):352-9; Vogler et al., 2001, Pediatr DevPathol. 4(5):421-33; Vogler et al., 2001, Pediatr Res. 49(3):342-8; andWolfe et al., 2000, Mol. Ther. 2(6):552-6), a mouse model formetachromatic leukodystrophy (see, e.g., Matzner et al., 2002, GeneTher. 9(1):53-63), a mouse model of Sandhoff disease (see, e.g., Sangoet al., 2002, Neuropathol Appl Neurobiol. 28(1):23-34), mouse models formucopolysaccharidosis type III A (see, e.g., Bhattacharyya et al., 2001,Glycobiology 11(1):99-10 and Bhaumik et al., 1999, Glycobiology9(12):1389-96.), arylsulfatase A (ASA)-deficient mice (see, e.g.,D'Hooge et al., 1999, Brain Res. 847(2):352-6 and D′Hooge et al, 1999,Neurosci Lett. 273(2):93-6); mice with an aspartylglucosaminuriamutation (see, e.g., Jalanko et al., 1998, Hum Mol. Genet. 7(2):265-72);feline models of mucopolysaccharidosis type VI (see, e.g., Crawley etal., 1998, J Clin Invest. 101(1):109-19 and Norrdin et al., 1995, Bone17(5):485-9); a feline model of Niemann-Pick disease type C (see, e.g.,March et al., 1997, Acta Neuropathol (Berl). 94(2):164-72); acidsphingomyelinase-deficient mice (see, e.g., Otterbach & Stoffel, 1995,Cell 81(7):1053-6), and bovine mannosidosis (see, e.g., Jolly et al.,1975, Birth Defects Orig Arctic Ser. 11(6):273-8).

Examples of animal models for tuberous sclerosis (“TSC”) include, butare not limited to, a mouse model of TSC1 (see, e.g., Kwiatkowski etal., 2002, Hum Mol. Genet. 11(5):525-34), a Tsc1 (TSC1 homologue)knockout mouse (see, e.g., Kobayashi et al., 2001, Proc Natl Acad SciUSA. 2001 Jul. 17; 98(15):8762-7), a TSC2 gene mutant (Eker) rat model(see, e.g., Hino 2000, Nippon Rinsho 58(6):1255-61; Mizuguchi et al.,2000, J Neuropathol Exp Neurol. 59(3):188-9; and Hino et al., 1999, ProgExp Tumor Res. 35:95-108); and Tsc2(+/−) mice (see, e.g., Onda et al.,1999, J Clin Invest. 104(6):687-95).

4.3 Synthesis and Preparation

The compounds of the invention can be obtained via standard, well-knownsynthetic methodology, see e.g. March, J. Advanced Organic Chemistry;Reactions Mechanisms, and Structure, 4th ed., 1992. Starting materialsuseful for preparing the compounds of the invention and intermediatestherefor, are commercially available or can be prepared fromcommercially available materials using known synthetic methods andreagents.

Compounds of Formulas I or II can be synthesized using the synthesisdepicted in schemes A and B below. The compounds of the presentinvention may be prepared by the methods discussed in the followingsection.

Compounds of formula I may be prepared using the methodology depicted inScheme A.

Commercially available, acid-labile resin A1 such as trityl resin,2-chlorotrityl chloride resin, phenylacetamidomethyl (PAM) resin, andp-alkoxybenzyl alcohol resin can be used in this invention. The couplingof benzoic acid compound A2 and trityl resin (here X=2-chlorotritylchloride) can be performed in a suitable solvent such asdichloromethane, dimethylformamide, toluene in the presence of atertiary amine reagent such as diisopropylethylamine or triethylamine.In an alternative method, the acylated resin A3 is conveniently preparedusing standard ester linkage formation conditions usingdiisopropylcarbodiimide (for phenylacetamidomethyl resin andp-alkoxybenzyl alcohol resin) or equivalents such asbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate(PyBOP), bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBrOP),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)without or with diisopropylethylamine in dimethylformamide. Theresin-bound cyanobenzoic ester can be treated with hydroxylamine in aninert solvent such as ethanol, tetrahydrofuran, dioxane anddimethylformamide or mixtures with or without diisopropylethylamine toafford the hydroxyamidine compound A4. The hydroxyamidine resin A4 canbe used as a common linker for the synthesis of 1,2,4-oxadiazole librarycompounds with variation of the rest of the compound of structure I asshown in Scheme A. The resin-bound hydroxyamidine compound is acylatedwith a reagent A5, wherein the group Y represents some leaving groups,such as halo, imidazoyl, p-nitrophenol, etc. in the presence of a basereagent, such as diisopropylethylamine or triethylamine, in an inertsolvent such as dichloromethane, tetrahydrofuran and dimethylformamideor mixtures. An alternative method, the acylation is convenientlycarried out with a reagent 5, wherein the group Y represents hydroxy,using diisopropylcarbodiimide or equivalents such asbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate,bromo-tris-pyrrolidino-phosphonium hexafluorophosphate,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride without orwith diisopropylethylamine in dimethylformamide. The resin-boundacylated compound A6 is cleaved under acidic conditions such as 2 molartrifluoroacetic acid in dichloromethane, or 3 molar acetic acid indichloromethane, to afford the desired compound A7. A ring-closurereaction on free acid compound A7 can be effected by the reflux in aninert solvent such as toluene, tetrahydrofuran, dioxane anddimethylformamide or mixtures with or without a base reagent such asdiisopropylethylamine, triethylamine or tetrabutylammonium fluoride toafford the 1,2,4-oxdiazole compound I. An alternative ring-closurereaction can be performed by dehydrocyclization of the resin-boundcompound A6 (Scheme A). This transformation can be accomplished with orwithout a base reagent such as triethylamine, diisopropylethylamine, ortetrabutylammonium fluoride in an inert solvent such as toluene,tetrahydrofuran, dioxane and dimethylformamide or mixtures. Temperaturesof the reaction range from ambient to reflux of the solvent.

The solid phase chemistry described above can be applied to the solutionphase synthesis of compounds of structure I. This is described in SchemeB, below.

The cyano compound B1 is hydroxyamidinated with hydroxylamine. Thisreaction is usually performed in the presence of a base reagent, such astriethyl amine, potassium carbonate or diisopropylethylamine, in asolvent such as methanol, ethanol, tert-butanol, tetrahydrofuran ordimethylformamide, and temperatures ranging from ambient to the refluxtemperature of the chosen solvent. The hydroxyamidine compound B2 isacylated with a reagent B3, wherein the group Y represents some leavinggroups, such as halo, imidazoyl, p-nitrophenol, etc. The reaction isusually carried out with a base reagent, such as triethyl amine ordiisopropylethylamine, in a solvent such as dichloromethane,tetrahydrofuran or dimethylformamide. An alternative method, theacylation is conveniently carried out under usual ester linkageformation reactions, wherein the group Y represents hydroxy, usingdiisopropylcarbodiimide or equivalents such asbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate,bromo-tris-pyrrolidino-phosphonium hexafluorophosphate,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride without orwith diisopropylethylamine. The ring-closure on the acylated compound B4can be accomplished with or without a base reagent such as triethylamine or diisopropylethylamine, in a solvent such as dichloromethane,tetrahydrofuran, toluene or dimethylformamide, and temperatures rangingfrom ambient to the reflux temperature of the chosen solvent.

4.4 Methods of Use

The invention encompasses methods of treating and preventing diseases ordisorders ameliorated by the suppression of premature translationtermination and/or nonsense-mediated mRNA decay in a patient whichcomprise administering to a patient in need of such treatment orprevention a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable prodrug, solvate,metabolite, polymorph, salt, solvate, hydrate, or clathrate thereof.

In one embodiment, the present invention encompasses the treatment orprevention of any disease that is associated with a gene exhibitingpremature translation termination and/or nonsense-mediated mRNA decay.In one embodiment, the disease is due, in part, to the lack ofexpression of the gene resulting from a premature stop codon. Specificexamples of genes which may exhibit premature translation terminationand/or nonsense-mediated mRNA decay and diseases associated withpremature translation termination and/or nonsense-mediated mRNA decayare found in U.S. Patent Application No. 60/390,747, titled: Methods ForIdentifying Small Molecules That Modulate Premature TranslationTermination And Nonsense Mediated mRNA Decay, filed Jun. 21, 2002, whichis incorporated herein by reference in its entirety.

Diseases ameliorated by the suppression of premature translationtermination and/or nonsense-mediated mRNA decay include, but are notlimited to: a genetic disease, cancer, an autoimmune disease, a blooddisease, a collagen disease, diabetes, a neurodegenerative disease, aproliferative disease, a cardiovascular disease, a pulmonary disease, aninflammatory disease or central nervous system disease.

Specific genetic diseases within the scope of the methods of theinvention include, but are not limited to, amyloidosis, hemophilia,Alzheimer's disease, Tay Sachs disease, atherosclerosis, giantism,dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, Parkinson'sdisease, Niemann Pick's disease, cystic fibrosis, muscular dystrophy,heart disease, kidney stones, ataxia-telangiectasia, familialhypercholesterolemia, retinitis pigmentosa, lysosomal storage disease,tuberous sclerosis, Duchenne muscular dystrophy, and Marfan syndrome.Both solid tumor and other cancers are included within the methods ofthe invention.

In another embodiment, the genetic disease is an autoimmune disease. Ina preferred embodiment, the autoimmune disease is rheumatoid arthritisor graft versus host disease.

In another embodiment, the genetic disease is a blood disease. In apreferred embodiment, the blood disease is hemophilia, Von Willebranddisease, ataxia-telangiectasia, b-thalassemia or kidney stones.

In another embodiment, the genetic disease is a collagen disease. In aembodiment, the collagen disease is osteogenesis imperfecta orcirrhosis.

In another embodiment, the genetic disease is diabetes.

In another embodiment, the genetic disease is an inflammatory disease.In a preferred embodiment, the inflammatory disease is arthritis.

In another embodiment, the genetic disease is a central nervous systemdisease. In one embodiment the central nervous system disease is aneurodegenerative disease. In a preferred embodiment, the centralnervous system disease is multiple sclerosis, muscular dystrophy,Duchenne muscular dystrophy, Alzheimer's disease, Tay Sachs disease,late infantile neuronal ceroid lipofuscinosis (LINCL) or Parkinson'sdisease.

In another embodiment, the genetic disease is cancer. In a preferredembodiment, the cancer is of the head and neck, eye, skin, mouth,throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach,prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine,heart or adrenals.

In another preferred embodiment, the cancer is associated with tumorsuppressor genes (see e.g. Garinis et al. 2002, Hum Gen 111:115-117;Meyers et al. 1998, Proc. Natl. Acad. Sci. USA, 95: 15587-15591; Kung etal. 2000, Nature Medicine 6(12): 1335-1340. Such tumor suppressor genesinclude, but are not limited to, APC, ATM, BRAC1, BRAC2, MSH1, pTEN, Rband p53.

In a particularly preferred embodiment, the tumor suppressor gene is thep53 gene. Nonsense mutations have been identified in the p53 gene andhave been implicated in cancer. Several nonsense mutations in the p53gene have been identified (see, e.g., Masuda et al., 2000, Tokai J ExpClin Med. 25(2):69-77; Oh et al., 2000, Mol Cells 10(3):275-80; Li etal., 2000, Lab Invest. 80(4):493-9; Yang et al., 1999, Zhonghua ZhongLiu Za Zhi 21(2):114-8; Finkelstein et al., 1998, Mol. Diagn.3(1):37-41; Kajiyama et al., 1998, Dis Esophagus. 11(4):279-83; Kawamuraet al., 1999, Leuk Res. 23(2):115-26; Radig et al., 1998, Hum Pathol.29(11):1310-6; Schuyer et al., 1998, Int J Cancer 76(3):299-303;Wang-Gohrke et al., 1998, Oncol Rep. 5(1):65-8; Fulop et al., 1998, JReprod Med. 43(2):119-27; Ninomiya et al., 1997, J Dermatol Sci.14(3):173-8; Hsieh et al., 1996, Cancer Lett. 100(1-2):107-13; Rall etal., 1996, Pancreas. 12(1):10-7; Fukutomi et al., 1995, Nippon Rinsho.53(11):2764-8; Frebourg et al., 1995, Am J Hum Genet. 56(3):608-15; Doveet al., 1995, Cancer Surv. 25:335-55; Adamson et al., 1995, Br JHaematol. 89(1):61-6; Grayson et al., 1994, Am J Pediatr Hematol Oncol.16(4):341-7; Lepelley et al., 1994, Leukemia. 8(8):1342-9; McIntyre etal., 1994, J Clin Oncol. 12(5):925-30; Horio et al., 1994, Oncogene.9(4):1231-5; Nakamura et al., 1992, Jpn J Cancer Res. 83(12):1293-8;Davidoff et al., 1992, Oncogene. 7(1):127-33; and Ishioka et al., 1991,Biochem Biophys Res Commun. 177(3):901-6; the disclosures of which arehereby incorporated by reference in their entireties). Any diseaseassociated with a p53 gene encoding a premature translation codonincluding, but not limited to, the nonsense mutations described in thereferences cited above, can be treated or prevented by compounds offormula I or II without being limited by theory these compounds mediatepremature translation termination and/or nonsense-mediated mRNA decay.

In other embodiments, diseases to be treated or prevented byadministering to a patient in need thereof an effective amount of acompound of formula I include solid tumor, sarcoma, carcinomas,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, a blood-born tumor, acutelymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute monoblastic leukemia, acuteerythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyelomonocytic leukemia, acute nonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, or multiple myeloma. Seee.g., Harrison's Principles of Internal Medicine, Eugene Braunwald etal., eds., pp. 491-762 (15th ed. 2001).

In a preferred embodiment, the invention encompasses a method oftreating or preventing a disease ameliorated by modulation of prematuretranslation termination and/or nonsense-mediated mRNA decay, orameliorating one or more symptoms associated therewith comprisingcontacting a cell with an effective amount of a compound of formula I orII. Cells encompassed by the present methods include animal cells,mammalian cells, bacterial cells, plant cells and virally infectedcells. In one embodiment, the nonsense codon was present in theprogenitor DNA. In another embodiment, the nonsense codon resulted frommutagenesis.

In certain embodiments, a compound of formula I or II, or apharmaceutically acceptable salt thereof, is administered to a patient,preferably a mammal, more preferably a human, as a preventative measureagainst a disease associated with premature translation terminationand/or nonsense-mediated mRNA decay.

In a preferred embodiment, it is first determined that the patient issuffering from a disease associate with premature translationtermination and/or nonsense-mediated mRNA decay. In another embodiment,the patient has undergone a screening process to determine the presenceof a nonsense mutation comprising the steps of screening a subject, orcells extracted therefrom, by an acceptable nonsense mutation screeningassay. In a preferred embodiment, the DNA of the patient can besequenced or subject to Southern Blot, polymerase chain reaction (PCR),use of the Short Tandem Repeat (STR), or polymorphic length restrictionfragments (RFLP) analysis to determine if a nonsense mutation is presentin the DNA of the patient. Alternatively, it can be determined ifaltered levels of the protein with the nonsense mutation are expressedin the patient by western blot or other immunoassays. In anotherembodiment, the patient is an unborn child who has undergone screeningin utero for the presence of a nonsense mutation. Administration of acompound of formula I or II can occur either before or after birth. In arelated embodiment, the therapy is personalized in that the patient isscreened for a nonsense mutation screening assay and treated by theadministration of one or more compounds of the invention; particularly,the patient may be treated with a compound particularly suited for themutations in question; e.g., depending upon the disease type, cell type,and the gene in question. Such methods are well known to one of skill inthe art.

In another embodiment, the cells (e.g., animal cells, mammalian cells,bacterial cells, plant cells and virally infected cells) are screenedfor premature translation termination and/or nonsense-mediated mRNAdecay with a method such as that described above (i.e., the DNA of thecell can be sequenced or subjected to Southern Blot, polymerase chainreaction (PCR), use of the Short Tandem Repeat (STR), or polymorphiclength restriction fragments (RFLP) analysis to determine if a nonsensemutation is present in the DNA of the cell).

Specific methods of the invention further comprise the administration ofan additional therapeutic agent (i.e. a therapeutic agent other than acompound of the invention). In certain embodiments of the presentinvention, the compounds of the invention can be used in combinationwith at least one other therapeutic agent. Therapeutic agents include,but are not limited to non-opioid analgesics; non-steroidanti-inflammatory agents; antiemetics; β-adrenergic blockers;anticonvulsants; antidepressants; Ca²⁺-channel blockers; anticanceragent and mixtures thereof.

In certain embodiments, the compounds of formula I or II can beadministered or formulated in combination with anticancer agents.Suitable anticancer agents include, but are not limited to, alkylatingagents; nitrogen mustards; folate antagonists; purine antagonists;pyrimidine antagoinists; spindle poisons; topoisomerase inhibitors;apoptosis inducing agents; angiogenesis inhibitors; podophyllotoxins;nitrosoureas; cisplatin; carboplatin; interferon; asparginase;tamoxifen; leuprolide; flutamide; megestrol; mitomycin; bleomycin;doxorubicin; irinotecan and taxol.

In certain embodiments, the compounds of formula I or II can beadministered or formulated in combination with antibiotics. In certainembodiments, the antibiotic is a macrolide (e.g., tobramycin (Tobi®)), acephalosporin (e.g., cephalexin (Keflex®), cephradine (Velosef®),cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime(Suprax®) or cefadroxil (Durice®)), a clarithromycin (e.g.,clarithromycin (Biaxin®)), an erythromycin (e.g., erythromycin(EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen VeeK®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin (Cipro®)or norfloxacin (Noroxin®)). In a preferred embodiment, the antibiotic isactive against Pseudomonas aeruginosa.

The compounds of the invention and the other therapeutics agent can actadditively or, more preferably, synergistically. In a preferredembodiment, a composition comprising a compound of the invention isadministered concurrently with the administration of another therapeuticagent, which can be part of the same composition or in a differentcomposition from that comprising the compounds of the invention. Inanother embodiment, a compound of the invention is administered prior toor subsequent to administration of another therapeutic agent.

The magnitude of a prophylactic or therapeutic dose of a particularactive ingredient of the invention in the acute or chronic management ofa disease or condition will vary, however, with the nature and severityof the disease or condition, and the route by which the activeingredient is administered. The dose, and perhaps the dose frequency,will also vary according to the age, body weight, and response of theindividual patient. Suitable dosing regimens can be readily selected bythose skilled in the art with due consideration of such factors. Ingeneral, the recommended daily dose range for the conditions describedherein lie within the range of from about 0.1 mg to about 2000 mg perday, given as a single once-a-day dose, preferably as divided dosesthroughout a day. In one embodiment, the daily dose is administered in asingle dose or in equally divided doses. Specifically, a daily doserange should be from about 5 mg to about 500 mg per day, morespecifically, between about 10 mg and about 200 mg per day. In managingthe patient, the therapy should be initiated at a lower dose, perhapsabout 1 mg to about 25 mg, and increased if necessary up to about 200 mgto about 2000 mg per day as either a single dose or divided doses,depending on the patient's global response.

It may be necessary to use dosages of the active ingredient outside theranges disclosed herein in some cases, as will be apparent to those ofordinary skill in the art. Furthermore, it is noted that the clinicianor treating physician will know how and when to interrupt, adjust, orterminate therapy in conjunction with individual patient response.

The phrases “therapeutically effective amount”, “prophylacticallyeffective amount” and “therapeutically or prophylactically effectiveamount,” as used herein encompass the above described dosage amounts anddose frequency schedules. Different therapeutically effective amountsmay be applicable for different diseases and conditions, as will bereadily known by those of ordinary skill in the art. Similarly, amountssufficient to treat or prevent such diseases, but insufficient to cause,or sufficient to reduce, adverse effects associated with conventionaltherapies are also encompassed by the above described dosage amounts anddose frequency schedules.

4.5 Pharmaceutical Compositions

Pharmaceutical compositions and single unit dosage forms comprising acompound of the invention, or a pharmaceutically acceptable polymorph,prodrug, salt, solvate, hydrate, or clathrate thereof, are alsoencompassed by the invention. Individual dosage forms of the inventionmay be suitable for oral, mucosal (including sublingual, buccal, rectal,nasal, or vaginal), parenteral (including subcutaneous, intramuscular,bolus injection, intraarterial, or intravenous), transdermal, or topicaladministration.

Pharmaceutical compositions and dosage forms of the invention comprise acompound of the invention, or a pharmaceutically acceptable prodrug,polymorph, salt, solvate, hydrate, or clathrate thereof. Pharmaceuticalcompositions and dosage forms of the invention typically also compriseone or more pharmaceutically acceptable excipients.

A particular pharmaceutical composition encompassed by this embodimentcomprises a compound of the invention, or a pharmaceutically acceptablepolymorph, prodrug, salt, solvate, hydrate, or clathrate thereof, and atleast one additional therapeutic agent. Examples of additionaltherapeutic agents include, but are not limited to: anti-cancer drugsand anti-inflammation therapies including, but not limited to, thoselisted above in Section 4.3.

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: tablets; caplets;capsules, such as soft elastic gelatin capsules; cachets; troches;lozenges; dispersions; suppositories; ointments; cataplasms (poultices);pastes; powders; dressings; creams; plasters; solutions; patches;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of inflammation or a related disease may containlarger amounts of one or more of the active ingredients it comprisesthan a dosage form used in the chronic treatment of the same disease.Similarly, a parenteral dosage form may contain smaller amounts of oneor more of the active ingredients it comprises than an oral dosage formused to treat the same disease or disorder. These and other ways inwhich specific dosage forms encompassed by this invention will vary fromone another will be readily apparent to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing,Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore carriers, excipients or diluents. Suitable excipients are wellknown to those skilled in the art of pharmacy, and non-limiting examplesof suitable excipients are provided herein. Whether a particularexcipient is suitable for incorporation into a pharmaceuticalcomposition or dosage form depends on a variety of factors well known inthe art including, but not limited to, the way in which the dosage formwill be administered to a patient. For example, oral dosage forms suchas tablets may contain excipients not suited for use in parenteraldosage forms. The suitability of a particular excipient may also dependon the specific active ingredients in the dosage form.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention comprise acompound of the invention, or a pharmaceutically acceptable salt,solvate, clathrate, hydrate, polymorph or prodrug thereof lie within therange of from about 0.1 mg to about 2000 mg per day, given as a singleonce-a-day dose in the morning but preferably as divided dosesthroughout the day taken with food. More specifically, the daily dose isadministered twice daily in equally divided doses. Specifically, a dailydose range should be from about 5 mg to about 500 mg per day, morespecifically, between about 10 mg and about 200 mg per day. In managingthe patient, the therapy should be initiated at a lower dose, perhapsabout 1 mg to about 25 mg, and increased if necessary up to about 200 mgto about 2000 mg per day as either a single dose or divided doses,depending on the patient's global response.

4.5.1 Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredient(s) in an intimate admixture with at least oneexcipient according to conventional pharmaceutical compoundingtechniques. Excipients can take a wide variety of forms depending on theform of preparation desired for administration. For example, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Because of their ease of administration, tablets and capsules representan advantageous oral dosage unit form, in which case solid excipientsare employed. If desired, tablets can be coated by standard aqueous ornonaqueous techniques. Such dosage forms can be prepared by any of themethods of pharmacy. In general, pharmaceutical compositions and dosageforms are prepared by uniformly and intimately admixing the activeingredients with liquid carriers, finely divided solid carriers, orboth, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,specifically from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, pre-gelatinized starch, otherstarches, clays, other algins, other celluloses, gums, and mixturesthereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

4.5.2 Delayed Release Dosage Forms

Active ingredients of the invention can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548,5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which isincorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active ingredients of the invention. The invention thusencompasses single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

4.5.3 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention.

4.5.4 Transdermal and Topical Dosage Forms

Transdermal and topical dosage forms of the invention include, but arenot limited to, creams, lotions, ointments, gels, solutions, emulsions,suspensions, or other forms known to one of skill in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, EastonPa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed.,Lea & Febiger, Philadelphia (1985). Transdermal dosage forms include“reservoir type” or “matrix type” patches, which can be applied to theskin and worn for a specific period of time to permit the penetration ofa desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal and topical dosage formsencompassed by this invention are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form lotions, tinctures, creams, emulsions, gelsor ointments, which are non-toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa. (1990).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients of the invention. For example, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water-soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

4.5.5 Mucosal Dosage Forms

Mucosal dosage forms of the invention include, but are not limited to,ophthalmic solutions, sprays and aerosols, or other forms known to oneof skill in the art. See, e.g., Remington's Pharmaceutical Sciences,18th eds., Mack Publishing, Easton Pa. (1990); and Introduction toPharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia(1985). Dosage forms suitable for treating mucosal tissues within theoral cavity can be formulated as mouthwashes or as oral gels. In oneembodiment, the aerosol comprises a carrier. In another embodiment, theaerosol is carrier free.

A compound of formula I or II can also be administered directly to thelung by inhalation (see e.g., Tong et al., PCT Application, WO 97/39745;Clark et al, PCT application, WO 99/47196, which are herein incorporatedby reference). For administration by inhalation, a compound of formula Ior II can be conveniently delivered to the lung by a number of differentdevices. For example, a Metered Dose Inhaler (“MDI”) which utilizescanisters that contain a suitable low boiling propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas can beused to deliver a compound of formula I directly to the lung. MDIdevices are available from a number of suppliers such as 3M Corporation,Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome,Schering Plough and Vectura.

Alternatively, a Dry Powder Inhaler (DPI) device can be used toadminister a compound of formula I to the lung (See, e.g., Raleigh etal., Proc. Amer. Assoc. Cancer Research Annual Meeting, 1999, 40, 397,which is herein incorporated by reference). DPI devices typically use amechanism such as a burst of gas to create a cloud of dry powder insidea container, which can then be inhaled by the patient. DPI devices arealso well known in the art and can be purchased from a number of vendorswhich include, for example, Fisons, Glaxo-Wellcome, Inhale TherapeuticSystems, ML Laboratories, Qdose and Vectura. A popular variation is themultiple dose DPI (“MDDPI”) system, which allows for the delivery ofmore than one therapeutic dose. MDDPI devices are available fromcompanies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough,SkyePharma and Vectura. For example, capsules and cartridges of gelatinfor use in an inhaler or insufflator can be formulated containing apowder mix of the compound and a suitable powder base such as lactose orstarch for these systems.

Another type of device that can be used to deliver a compound of formulaI or II to the lung is a liquid spray device supplied, for example, byAradigm Corporation. Liquid spray systems use extremely small nozzleholes to aerosolize liquid drug formulations that can then be directlyinhaled into the lung.

In a preferred embodiment, a nebulizer device is used to deliver acompound of formula I or II to the lung. Nebulizers create aerosols fromliquid drug formulations by using, for example, ultrasonic energy toform fine particles that can be readily inhaled (See e.g., Verschoyle etal., British J Cancer, 1999, 80, Suppl 2, 96, which is hereinincorporated by reference). Examples of nebulizers include devicessupplied by Sheffield/Systemic Pulmonary Delivery Ltd. (See, Armer etal., U.S. Pat. No. 5,954,047; van der Linden et al., U.S. Pat. No.5,950,619; van der Linden et al., U.S. Pat. No. 5,970,974, which areherein incorporated by reference), Aventis and Batelle PulmonaryTherapeutics. Inhaled compound of formula I, delivered by nebulizerdevices, is currently under investigation as a treatment foraerodigestive cancer (Engelke et al., Poster 342 at American Associationof Cancer Research, San Francisco, Calif., Apr. 1-5, 2000) and lungcancer (Dahl et al., Poster 524 at American Association of CancerResearch, San Francisco, Calif., Apr. 1-5, 2000).

In a particularly preferred embodiment, an electrohydrodynamic (“EHD”)aerosol device is used to deliver a compound of formula I or II to thelung. MID aerosol devices use electrical energy to aerosolize liquiddrug solutions or suspensions (see e.g., Noakes et al., U.S. Pat. No.4,765,539; Coffee, U.S. Pat. No. 4,962,885; Coffee, PCT Application, WO94/12285; Coffee, PCT Application, WO 94/14543; Coffee, PCT Application,WO 95/26234, Coffee, PCT Application, WO 95/26235, Coffee, PCTApplication, WO 95/32807, which are herein incorporated by reference).The electrochemical properties of the compound of formula I formulationmay be important parameters to optimize when delivering this drug to thelung with an EHD aerosol device and such optimization is routinelyperformed by one of skill in the art. EHD aerosol devices may moreefficiently delivery drugs to the lung than existing pulmonary deliverytechnologies. Other methods of intra-pulmonary delivery of a compound offormula I or II will be known to the skilled artisan and are within thescope of the invention.

Liquid drug formulations suitable for use with nebulizers and liquidspray devices and EHD aerosol devices will typically include a compoundof formula I with a pharmaceutically acceptable carrier. Preferably, thepharmaceutically acceptable carrier is a liquid such as alcohol, water,polyethylene glycol or a perfluorocarbon. Optionally, another materialmay be added to alter the aerosol properties of the solution orsuspension of a compound of formula I or II. Preferably, this materialis liquid such as an alcohol, glycol, polyglycol or a fatty acid. Othermethods of formulating liquid drug solutions or suspension suitable foruse in aerosol devices are known to those of skill in the art (See,e.g., Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No.5,556,611, which are herein incorporated by reference) A compound offormula I can also be formulated in rectal or vaginal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, a compound offormula I or II can also be formulated as a depot preparation. Such longacting formulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Alternatively, other pharmaceutical delivery systems can be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat can be used to deliver a compound of formula I or II. Certainorganic solvents such as dimethylsulfoxide can also be employed,although usually at the cost of greater toxicity. A compound of formulaI can also be delivered in a controlled release system. In oneembodiment, a pump can be used (Sefton, CRC Crit. Ref Biomed Eng., 1987,14, 201; Buchwald et al., Surgery, 1980, 88, 507; Saudek et al., N.Engl. J. Med, 1989, 321, 574). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, J Macromol. Sci.Rev. Macromol. Chem., 1983, 23, 61; see also Levy et al., Science 1985,228, 190; During et al., Ann. Neurol., 1989, 25, 351; Howard et al.,1989, J. Neurosurg. 71, 105). In yet another embodiment, acontrolled-release system can be placed in proximity of the target ofthe compounds of the invention, e.g., the lung, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115 (1984)).Other controlled-release system can be used (see e.g. Langer, Science,1990, 249, 1527).

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide mucosal dosage forms encompassed by thisinvention are well known to those skilled in the pharmaceutical arts,and depend on the particular site or method which a given pharmaceuticalcomposition or dosage form will be administered. With that fact in mind,typical excipients include, but are not limited to, water, ethanol,ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, mineral oil, and mixtures thereof, which arenon-toxic and pharmaceutically acceptable. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa. (1990).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, canalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

5. EXAMPLES

The following examples employ methodology which can be used to prepareall of the compounds embodied in this invention, provided theappropriate reagents and substrates are utilized, and minor variationsof the described conditions are maintained. Such variations would beeasily performed by one of skill in the art without undueexperimentation given the description below.

5.1 Example 1 Preparation of3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid

40 g of 2-chlorotrityl chloride resin (Rapp polymere, Germany), wassuspended in dry dimethylformamide (200 mL) for 10 min and the solventwas drained. To the resin was added a solution of 3-cyanobenzoic acid(12.71 g, 96.4 mmol) in 300 mL of dimethylformamide and agitated 4 h atroom temperature. The solvents were drained and the resin was washedwith dichloromethane (3×200 mL×1 min), dimethylformamide (3×200 mL×1min), methanol (3×200 mL×1 min), and dichloromethane (3×200 mL×1 min).The resin was vacuum dried for 4 h. The desired product was analyzed bycleavage of a small amount of the reacted resin withtriethylsilane/trifluoroacetic acid/dichloromethane (10/50/40). LC/MS(ESI) m/z 148 [M+H]⁺ and 97% purity.

The 3-cyanobenzoic trityl resin in ethanol (300 mL) was agitated for 10min at room temperature, and then the solvent was drained. To a solutionof hydroxy amine hydrochloride (35.81 g, 516 mmol) in ethanol (200 mL)was added diisopropylethylamine (89.3 mL, 516 mmol) and stirred 5 min atroom temperature. To the resin was added the reaction mixture andagitated 24 h at 40° C. The solvents were drained, and the resin waswashed with dichloromethane (3×200 mL×10 min), dimethylformamide (3×200mL×10 min), methanol (3×200 mL×10 min), and dichloromethane (3×200 mL×10min). The resin was vacuum dried for 4 h. The desired product wasanalyzed by cleavage of a small amount of the reacted resin withtriethylsilane/trifluoroacetic acid/dichloromethane (10/50/40). LC/MS(ESI) m/z 181 [M+H]⁺ and 90% purity.

To a suspension of hydroxyamidine resin (500 mg, 0.4 mmol) in anhydrousdichloromethane (3 mL) was added 4-Fluorobenzoyl chloride (95 uL, 0.8mmol) and diisopropylethylamine (138 uL, 0.8 mmol). The reaction mixturewas agitated overnight at room temperature. The solvents were drained,and the resin was washed with dichloromethane (3×10 mL×10 min),dimethylformamide (3×10 mL×10 min), methanol (3×10 mL×10 min), anddichloromethane (3×10 mL×10 min). The resin was vacuum dried for 4 h.The desired product was analyzed by cleavage of a small amount of thereacted resin with triethylsilane/trifluoroacetic acid/dichloromethane(10/50/40). LC/MS (ESI) m/z 303 [M+H]⁺ and 65% purity.

To a suspension of acylated resin in anhydrous dichloromethane (1.5 mL)was added 50% trifluoroacetic acid in dichloromethane (1.5 mL). Thereaction mixture was agitated 2 h at room temperature. The resin wasremoved and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in 10% dimethylformamide in toluene (4 mL) andthen stirred for 2 h at 130° C. The solvents were removed and thedesired product was purified by preparative LC/MS. LC/MS (ESI) m/z 285[M+H]⁺ and 98% purity.

The following compounds are prepared using the procedures describedabove. Compounds are analyzed by a LC/MS using Electrospray ionization(ESI).

TABLE 2 Compound Compound Name [M + H]⁺

3-[5-(3-Chloro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 301.7

3-[5-(4-Pentyloxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 353.4

3-(5-Naphthalen-1-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 317.3

3-(5-p-Tolyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 281.3

3-(5-Biphenyl-4-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 343.3

3-[5-(4-Isobutyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 323.4

3-(5-Phenyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 267.3

3-(5-Cyclohexyl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 273.3

3-[5-(3,4,5-Trimethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid357.3

3-[5-(4-Nitro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 312.2

3-[5-(4-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 297.3

3-[5-(o-tolyl)-[1,2,4]oxadiazol-3- yl]-benzoic acid 281.3

3-(5-Benzo[1,3]dioxol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 311.3

3-(5-Isopropyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 233.2

3-[5-(3,5-Bis-trifluoromethyl- phenyl)-[1,2,4]oxadiazol-3-yl]- benzoicacid 403.2

3-[5-(4-Trifluoromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid335.2

3-[5-(4-Dimethylamino-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 310.3

3-[5-(2-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 297.3

3-[5-(3-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 297.3

3-(5-Furan-2-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 257.2

3-(5-tert-Butyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 247.3

3-(5-Benzo[1,2,5]oxadiazol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid309.2

3-[5-(4-Chloromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 315.7

3-[5-(4-tert-Butyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 323.4

3-(5-Butyl-[1,2,4]oxadiazol-3-yl)- benzoic acid 247.3

3-(5-Cyclopropyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 231.2

3-(5-Thiophen-2-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 273.3

3-(5-Propenyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 231.2

3-(5-Cyclopentyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 259.3

3-(5-Thiophen-2-ylmethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 287.3

3-[5-(4-Chloro-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 315.7

3-[5-(4-Chloro-phenoxymethyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 331.7

3-[5-(2-Trifluoromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid335.3

3-[5-(2,6-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2

3-[5-(4-Ethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 296.3

3-[5-(3,4-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 304.2

3-(5-m-Tolyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 281.3

3-[5-(4-Pyrrol-1-yl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 332.0

3-(5-Benzyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 281.3

3-(5-Methoxymethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 235.2

3-[5-(2,5-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2

3-[5-(1-Phenyl-propyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 309.3

3-[5-(2-Chloro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 301.7

3-[5-(3-Trifluoromethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid351.2

3-[5-(4-Fluoro-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 299.3

3-[5-(2,5-Dimethyl-furan-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid285.3

3-[5-(3-Methyl-thiophen-2-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 287.3

3-[5-(3-Chloro-phenoxymethyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 331.7

3-(5-Isoxazol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 258.2

3-[5-(6-Chloro-pyridin-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 302.7

3-{5-[3-(2-Chloro-phenyl)-5- methyl-isoxazol-4-yl]-[1,2,4]oxadiazol-3-yl}-benzoic acid 382.8

3-{5-[3-(2-Chloro-6-fluoro- phenyl)-5-methyl-isoxazol-4-yl]-[1,2,4]oxadiazol-3-yl}-benzoic acid 400.0

3-(5-Cyclopentylmethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 273.3

3-[5-(2,4-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2

3-(5-Pyridin-3-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 268.2

3-(5-Pyridin-4-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 268.2

3-(5-Cyclobutyl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 245.2

3-[5-(4-Methoxy-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 311.3

3-[5-(3,4-Dimethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 327.3

3-[5-(2-Chloro-pyridin-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 302.7

3-[5-(1-Acetyl-piperidin-4-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid316.3

3-(5-Benzo[b]thiophen-2-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 323.3

3-[5-(3-Dimethylamino-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 310.3

3-[5-(2,3-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2

3-[5-(2-Fluoro-5-methyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid299.3

3-[5-(2-Methylsulfanyl-pyridin-3- yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid 314.3

3-[5-(2,2-Difluoro- benzo[1,3]dioxol-5-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid 347.2

2-Fluoro-5-[5-(4-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid303.2

3-[5-(4-Bromo-2-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid364.1

3-[5-(3-Fluoro-biphenyl-4-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 361.3

3-[5-(3-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic aid 285.2

3-[5-(2,3-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic aid 303.2

3-[5-(2-Fluoro-5-methyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid299.3

3-[5-(2-Methylsulfanyl-pyridin-3- yl)-[1,2,4]oxadiazol-3-yl]-benzoicacid 314.3

3-[5-(2,2-Difluoro- benzo[1,3]dioxol-5-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid 347.2

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 285.1

3-[5-(4-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 285.2

3-[5-(4-Cyano-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 292.08

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid sodium salt306.04

3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester299.08

5.2 Example 2 Preparation of3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid

To a solution of 3-Cyanobenzoic acid (44.14 g, 300 mmol) in DMF (0.6 L)was added K₂CO₃ (62.19 g, 450 mmol) and then stirred for 30 min at roomtemperature. To the suspension was added methyl iodide (28 mL, 450 mmol)over 20 min, and the reaction mixture was stirred further 4 h at roomtemperature. The reaction mixture was poured to 1.2 L of ice water andstirred for 30 min, and the precipitate was filtered off. The white cakewas dissolved in methanol (70 mL), and then re-precipitated in coldwater. The desired product was obtained as a white powder with 79% yield(38 g, 99% purity by LC/UV). ¹H-NMR (CDCl₃) δ 8.85 (2H), 8.28 (1H), 8.02(1H), 4.17 (3H).

To a solution of 3-Cyanobenzoic acid methyl ester (50 g, 310 mmol) inethanol (500 mL) was added 50% aqueous hydroxylamine (41 mL, 620 mmol)at room temperature. The reaction mixture was stirred for 1 h at 100° C.and the solvents were removed under reduced pressure. The oily residuewas dissolved in 20/80 ethanol/toluene (50 mL×2) and then concentratedagain. The desired ester (61 g, quan. yield) was obtained as a whitepowder with 98% purity (LC/UV). ¹H-NMR (CDCl₃) δ 9.76 (1H), 8.24 (1H),7.82 (2H), 7.51 (1H), 5.92 (2H), 3.82 (3H).

To a solution of 3-(N-Hydroxycarbamimidoyl)-benzoic acid methyl ester(60 g, 310 mmol) in anhydrous THF (200 mL) was addeddiisopropylethylamine (75 mL, 434 mmol) at 5° C., and then to themixture was added 2-fluorobenzoyl chloride (48.1 mL, 403 mmol) over 20min. The reaction mixture was stirred for 1 h at room temperature. Theprecipitate was filtered off and the filtrate was concentrated underreduced pressure. The residue was dissolved in ethylacetate (400 mL) andthen washed with water (200 mL×2). The solvent was removed under reducedpressure and the desired product was crystallized in 60% ethylacetate inhexane to yield the desired product (81 g, 83% yield) as a white solid.¹H-NMR (CDCl₃) δ 8.18 (1H), 8.03 (2H), 7.48 (2H), 7.18 (2H), 5.61 (2H),3.82 (31-1).

44 g of 3-(N-2-Fluorobenzoylcarbamimidoyl)-benzoic acid methyl ester intoluene (500 mL) was refluxed for 4 h at 130° C. using Dean-Starkapparatus. The reaction mixture was stirred at 5° C. for 18 h. The whiteprecipitate was filtered off and the filtrate was concentrated,crystallized again in toluene. The desired oxadiazole (38 g, 92% yield)was obtained as a white solid with 99% purity (LC/UV). ¹H-NMR (CDCl₃) δ8.91 (1H), 8.38 (1H), 8.15 (2H), 7.62 (2H), 7.35 (2H), 3.95 (3H)

To a solution of 3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid methyl ester (33 g, 111 mol) in TI-IF (400 mL) was added 1.5Maqueous NaOH (100 mL, 144 mmol). The reaction mixture was refluxed for 2h at 100° C. The organic solvent was removed under reduced pressure andthe aqueous solution was stirred 2 h at 5° C. The white precipitate wasfiltered off and the filtrate was concentrated and precipitated again inwater. The white cake was washed with cold water and then dried usinglyophilizer. The desired salt (33 g, 96% yield) was obtained as a whitepowder with 98.6% purity (LC/UV).

To a solution of 3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid methyl ester (3.3 g, 11 mmol) in THF (40 mL) was added 1.5M aqueousNaOH (10 mL, 14 mmol). The reaction mixture was refluxed for 2 h at 100°C. The organic solvent was removed and the aqueous solution was dilutedwith water (50 mL), and then acidified with aqueous HCl. The whiteprecipitate was filtered off and the white cake was washed with coldwater and then dried using lyophilizer. The desired acid (3.0 g, 96%yield) was obtained as a white powder with 98% purity (LC/UV). Meltingpoint 242° C.; IR ν 3000 (Aromatic C—H), 1710 (C═O); ¹H-NMR (D₆-DMSO) δ8.31 (1H), 8.18 (2H), 8.08 (1H), 7.88 (2H), 7.51 (2H); ¹³C-NMR (D₆-DMSO)δ 172.71, 167.38, 166.48, 161.25, 135.80, 132.24, 131.79, 131.79,131.08, 130.91, 129.81, 127.76, 125.48, 117.38, 111.70; ¹⁹F-NMR(D₆-DMSO) δ 109.7.

The following compounds are prepared using the procedures describedabove.

TABLE 3 Compound Compound Name [M + H]⁺

3-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 285.2

3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 285.1

3-[5-(3-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 285.2

4-Fluoro-3-[5-(4- fluoro-phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid303.2

5-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]-2-methoxy- benzoic acid315.3

3-[5-(4-Chloro-2- fluoro-phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid319.7

3-[5-(3-Fluoro- biphenyl-4-yl)- [1,2,4]oxadiazol-3- yl]-benzoic acid361.3

3-[5-(4-Bromo-2- fluoro-phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid364.1

3-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid methylester 299.08

5-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-2-methoxy- benzoic acid339.13

3-[5-(4-Bromo-2- fluoro-phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid365.05

3-[5-(3-Fluoro- biphenyl-4-yl)- [1,2,4]oxadiazol-3- yl]-benzoic acid361.16

3-[5-(6-Pyrrolidin-1- yl-pyridin-3-yl)- [1,2,4]oxadiazol-3- yl]-benzoicacid 337.20

3-[5-(6-Morpholin-4- yl-pyridin-3-yl)- [1,2,4]oxadiazol-3- yl]-benzoicacid 353.18

3-[5-(3,4,5,6- Tetrahydro-2H- [1,2′]bipyridinyl-5′-yl)-[1,2,4]oxadiazol- 3-yl]-benzoic acid 351.18

3-[5-(2-Fluoro-6- hydroxy-phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid301.18

3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 2-methoxy-ethyl ester 343.16

3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 2-(2-methoxy-ethoxy)- ethyl ester 387.49

3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 2-[2-(2-methoxy- ethoxy)-ethoxy]- ethyl ester 431.31

3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 2-{2-[2-(2-methoxy- ethoxy)-ethoxy]- ethoxy}-ethyl ester 475.26

3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 2-(2-{2-[2-(2-methoxy- ethoxy)-ethoxy]- ethoxy}-ethoxy)- ethyl ester519.33

3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 2-[2-(2-{2-[2-(2- hydroxy-ethoxy)- ethoxy]-ethoxy}- ethoxy)-ethoxy]- ethylester 549.35

3-[5-(4-Amino- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 282.20

3-[5-(4-Azido- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 309.20

3-[5-(4-Benzyloxy- phenyl)- [1,2,4]oxadiazol-3- yl]-benzoic acid 373.16

5.3 Example 3 Identification and Characterization of Compounds thatPromote Nonsense Suppression and/or Modulate Translation Termination

The assays described above in Section 4.2 were used in two highthroughput screens. Compounds were screened in the cell-based andbiochemical assays. Compounds were tested, resynthesized and testedagain to confirm chemical structures.3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodiumsalt was characterized further with the in vitro luciferase nonsensesuppression assay. To ensure that the observed nonsense suppressionactivity of the selected compounds was not limited to the rabbitreticulocyte assay system, HeLa cell extract was prepared and optimized(Lie & Macdonald, 1999, Development 126(22):4989-4996 and Lie &Macdonald, 2000, Biochem. Biophys. Res. Commun. 270(2):473-481).

5.4 Example 4 Characterization of Compounds that Increase NonsenseSuppression and Produce Functional Protein

It was previously demonstrated that compounds of the invention increasethe level of nonsense suppression in the biochemical assay three to fourfold over untreated extracts. To determine whether compounds alsofunction in vivo, a stable cell line harboring the UGAnonsense-containing luciferase gene was treated with selected compounds.Cells were grown in standard medium supplemented with 1%penicillin-streptomycin (P/S) and 10% fetal bovine serum (FBS) to 70%confluency and split 1:1 the day before treatment. On the following day,cells were trypsinized and 40,000 cells were added to each well of a96-well tissue culture dish. Serial dilutions of each compound wereprepared to generate a six-point dose response curve spanning 2 logs (30μM to 0.3 μM). The final concentration of the DMSO solvent remainedconstant at 1% in each well. Cells treated with 1% DMSO served as thebackground standard, and cells treated with gentamicin served as apositive control.

5.5 Example 5 3-[2-(4-Isopropyl-3-methyl phenoxy)-acetylamino]-benzoicacid, Sodium Salt Alters the Accessibility of the Chemical ModifyingAgents to Specific Nucleotides in the 28 S rRNA

Previous studies have demonstrated that gentamicin and other members ofthe aminoglycoside family that decrease the fidelity of translation bindto the A site of the 16S rRNA. By chemical footprinting, UVcross-linking and NMR, gentamicin has been shown to bind at the A site(comprised of nucleotides 1400-1410 and 1490-1500, E. coli numbering) ofthe rRNA at nucleotides 1406, 1407, 1494, and 1496 (Moazed & Noller,1987, Nature 327(6121):389-394; Woodcock et al., 1991, EMBO J.10(10):3099-3103; and Schroeder et al., 2000, EMBO J. 19:1-9

Ribosomes prepared from HeLa cells were incubated with the smallmolecules (at a concentration of 100 μM), followed by treatment withchemical modifying agents (dimethyl sulfate [DMS] and kethoxal [KE]).Following chemical modification, rRNA was phenol-chloroform extracted,ethanol precipitated, analyzed in primer extension reactions usingend-labeled oligonucleotides hybridizing to different regions of thethree rRNAs and resolved on 6% polyacrylamide gels. The probes used forprimer extension cover the entire 18S (7 oligonucleotide primers), 28S(24 oligonucleotide primers), and 5S (one primer)_(r)RNAs. Controls inthese experiments include DMSO (a control for changes in rRNAaccessibility induced by DMSO), paromomycin (a marker for 18S rRNAbinding), and anisomycin (a marker for 28S rRNA binding).

The results of these foot-printing experiments indicated that3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodiumsalt alters the accessibility of the chemical modifying agents tospecific nucleotides in the 28S rRNA. More specifically, the regionsprotected by 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoicacid, sodium salt include: (1) a conserved region in the vicinity of thepeptidyl transferase center (domain V) implicated in peptide bondformation and (2) a conserved region in domain II that may interact withthe peptidyl transferase center based on binding of vernamycinin B toboth these areas.

5.6 Example 6 Readthrough of Premature Termination Codons in Cell-basedDisease Models

To address the effects of the nonsense-suppressing compounds on mRNAsaltered in specific inherited diseases, a bronchial epithelial cell lineharboring a nonsense codon at amino acid 1282 (W1282X) was treated with3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodiumsalt (20 μM) and CFTR function was monitored as a cAMP-activatedchloride channel using the SPQ assay (Yang et al., 1993, Hum Mol. Genet.2(8):1253-1261 and Howard et al., 1996, Nat. Med. 2(4):467-469). Theseexperiments showed that cAMP treatment of these cells resulted in anincrease in SPQ fluorescence, consistent with stimulation ofCFTR-mediated halide efflux. No increase in fluorescence was observedwhen cells were not treated with compound or if the cells were notstimulated with cAMP. These results indicate that the full-length CFTRexpressed from this nonsense-containing allele following compoundtreatment also functions as a cAMP-stimulated anion channel, thusdemonstrating that cystic fibrosis cell lines increase chloride channelactivity when treated with3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodiumsalt.

5.7 Example 7 Primary Cells from the Mdx Nonsense-containing MouseExpress Full-length Dystrophin Protein When Treated with3-[2-(4-Isopropyl-3-methyl-phenozy)-acetylamino]benzoic acid, SodiumSalt

The mutation in the mdx mouse that premature termination of the 427 kDadystrophin polypeptide has been shown to be a C to T transition atposition 3185 in exon 23 (Sicinski et al., 1989, Science.244(4912):1578-1580). Mouse primary skeletal muscle cultures derivedfrom 1-day old mdx mice were prepared as described previously(Barton-Davis et al., 1999, J Clin Invest. 104(4):375-381). Cells werecultured for 10 days in the presence of3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodiumsalt (20 μM). Culture medium was replaced every four days and thepresence of dystrophin in myoblast cultures was detected byimmunostaining as described previously (Barton-Davis et al., 1999, JClin Invest. 104(4):375-381). A primary monoclonal antibody to theC-terminus of the dystrophin protein (F19A12) was used undiluted andrhodamine conjugated anti-mouse IgG was used as the secondary antibody.The F19A12 antibody will detect the full-length protein produced bysuppression of the nonsense codon. Staining was viewed using a Leica DMRmicropscope, digital camera, and associated imaging software at theUniversity of Pennsylvania.

5.8 Example 8 Readthrough of Premature Termination Codons in the MdxMouse

As previously described (Barton-Davis et al., 1999, J Clin Invest.104(4):375-381), compound was delivered by Alzet osmotic pumps implantedunder the skin of anesthetized mice. Two doses of3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodiumsalt were administered. Gentamicin served as a positive control andpumps filled with solvent only served as the negative control. Pumpswere loaded with appropriate compound such that the calculated doses towhich tissue was exposed were 10 μM and 20 μM. The gentamicinconcentration was calculated to achieve tissue exposure of approximately200 μM. In the initial experiment, mice were treated for 14 days, afterwhich animals were anesthetized with ketamine and exsanguinated. Thetibialis anterior (TA) muscle of the experimental animals was thenexcised, frozen, and used for immunofluorescence analysis of dystrophinincorporation into striated muscle. The presence of dystrophin in TAmuscles was detected by immunostaining, as described previously(Barton-Davis et al., 1999, J Clin Invest. 104(4):375-381).

5.9 Example 9 200 Mg Dosage Capsule

Table 3 illustrates a batch formulation and single dosage formulationfor a 200 mg single dose unit, i.e., about 40 percent by weight.

TABLE 3 Formulation for 200 mg capsule Percent Quantity QuantityMaterial By Weight (mg/tablet) (kg/batch) Compound of the 40.0% 200 mg16.80 kg invention Pregelatinized Corn 9.5% 297.5 mg 24.99 kg Starch,NF5 Magnesium Stearate 0.5% 2.5 mg 0.21 kg Total 100.0% 500 mg 42.00 kg

The pregelatinized corn starch (SPRESS B-820) and compound of theinvention components are passed through a 710 μm screen and then areloaded into a Diffusion Mixer with a baffle insert and blended for 15minutes. The magnesium stearate is passed through a 210 μm screen and isadded to the Diffusion Mixer. The blend is then encapsulated in a size#0 capsule, 500 mg per capsule (8400 capsule batch size) using a Dosatortype capsule filling machine.

5.10 Example 10 100 Mg Oral Dosage Form

Table 4 illustrates a batch formulation and a single dose unitformulation containing 100 mg of a compound of the invention.

TABLE 4 Formulation for 100 mg tablet Percent Quantity Quantity Materialby Weight (mg/tablet) (kg/batch) compound of the  40% 100.00 20.00invention Microcrystalline 53.5%  133.75 26.75 Cellulose, NF PluronicF-68 4.0% 10.00 2.00 Surfactant Croscarmellose 2.0% 5.00 1.00 SodiumType A, NF Magnesium Stearate, 0.5% 1.25 0.25 NF Total 100.0%  250.00 mg50.00 kg

The microcrystalline cellulose, croscarmellose sodium, and compound ofthe invention components are passed through a #30 mesh screen (about430μ to about 655μ). The Pluronic F-68® (manufactured by JRHBiosciences, Inc. of Lenexa, Kans.) surfactant is passed through a #20mesh screen (about 457μ to about 1041μ). The Pluronic F-68® surfactantand 0.5 kgs of croscarmellose sodium are loaded into a 16 qt. twin shelltumble blender and are mixed for about 5 minutes. The mix is thentransferred to a 3 cubic foot twin shell tumble blender where themicrocrystalline cellulose is added and blended for about 5 minutes. Thecompound is added and blended for an additional 25 minutes. Thispre-blend is passed through a roller compactor with a hammer millattached at the discharge of the roller compactor and moved back to thetumble blender. The remaining croscarmellose sodium and magnesiumstearate is added to the tumble blender and blended for about 3 minutes.The final mixture is compressed on a rotary tablet press with 250 mg pertablet (200,000 tablet batch size).

5.11 Example 11 Aerosal Dosage Form

A concentrate is prepared by combining a compound of the invention, anda 12.6 kg portion of the trichloromonofluoromethane in a sealedstainless steel vessel equipped with a high shear mixer. Mixing iscarried out for about 20 minutes. The bulk suspension is then preparedin the sealed vessel by combining the concentrate with the balance ofthe propellants in a bulk product tank that is temperature controlled to21° to 27° C. and pressure controlled to 2.8 to 4.0 BAR. 17 ml aerosolcontainers that have a metered valve which is designed to provide 100inhalations of the composition of the invention. Each container isprovided with the following:

compound of the invention 0.0141 g trichloromonofluoromethane 1.6939 gdichlorodifluoromethane 3.7028 g dichlorotetrafluoroethane 1.5766 gtotal 7.0000 g

1. A pharmaceutical composition comprising a compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein: Z is substitutedaryl, substituted or unsubstituted cycloalkyl, substituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedarylalkyl; R¹ is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, —(CH₂CH₂O)_(n)R⁶ or any biohydrolyzable group; R², R³, R⁴ and R⁵are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl; substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, alkoxy, aryloxy, halogen, CF₃, OCF₃, OCHF₂, CN,COOH, COOR⁷, SO₂R⁷, NO₂, NH₂, or N(R⁷) ₂; R⁶ is hydrogen or substitutedor unsubstituted alkyl; each occurrence of R⁷ is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl; substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, alkoxy,aryloxy, halogen or CF₃; n is an integer from 1 to 7, with the provisothat when R¹, R², R³, R⁴ and R⁵ are each hydrogen, then Z is not2-carboxy ethyl, 3-(4-pyridinyl)propyl, 2-(4-piperidinyl) ethyl,4-cyano-phenyl or alkyl substituted with substituted carbamyl, and apharmaceutically acceptable carrier, excipient or diluent.
 2. Thepharmaceutical composition of claim 1, wherein Z is substituted aryl. 3.The pharmaceutical composition of claim 2, wherein substituted aryl ishalo substituted phenyl.
 4. A pharmaceutical composition comprising acompound of the formula:

or a pharmaceutically acceptable salt thereof, wherein: X is halogen,substituted alkyl, alkoxy or hydroxy; R¹ is hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl,—(CH₂CH₂O)_(n)R⁶ or any biohydrolyzable group; R², R³, R⁴ and R⁵ areindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl;substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, alkoxy, aryloxy, heteroaryloxy, halogen, CF₃,OCF₃, OCHF₂, CN, COOH, COOR⁷, SO₂R⁷, NO₂, NH₂, or N(R⁷) ₂; R⁶ ishydrogen or substituted or unsubstituted alkyl; each occurrence of R⁷ isindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl;substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, alkoxy, aryloxy, heteroaryloxy, halogen orCF₃; and n is an integer from 1 to 7, and a pharmaceutically acceptablecarrier, excipient or diluent.
 5. A pharmaceutical compositioncomprising a compound selected from:3-[5-(4-Chloromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3,4-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Ethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Chloro-phenyl-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-tert-Butyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,5-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,4-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-Chloro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,6-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Dimethylamino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Dimethylamino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3,5-Bis-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid; 3-[5-(3,4-Dimethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Pentyloxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3,4,5-Trimethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Isobutyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,3-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-5-methyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;4-Fluoro-3-[5-(4-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;2-Fluoro-5-[5-(4-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Chloro-2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Bromo-2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid sodium salt;3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester;5-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-2-methoxy-benzoic acid;3-[5-(2-Fluoro-6-hydroxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-methoxy-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-methoxy-ethoxy)-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-[2-(2-methoxy-ethoxy) -ethoxy]-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-[2-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethylester; 3-[5-(4-Amino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Azido-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Benzyloxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-p-Tolyl-[1,2,41]oxadiazol-3-yl)-benzoic acid;3-[5-(o-tolyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-m-Tolyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclohexyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclopropyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclopentyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclobutyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Thiophen-2-yl-methyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(4-Chloro-benzyl)-[1,2,41]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Chloro-phenoxymethyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-Benzyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Methoxymethyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(1-Phenyl-propyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Fluoro-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Chloro-phenoxymethyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-Cyclopentylmethyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(4-Methoxy-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid; and apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, excipient or diluent.
 6. The pharmaceuticalcomposition of claim 5, wherein the compound is3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof.
 7. The pharmaceuticalcomposition of claim 6, suitable for oral, parenteral, mucosal,transdermal or topical administration.
 8. The pharmaceutical compositionof claim 7, suitable for oral administration.
 9. The pharmaceuticalcomposition of claim 8, in solid form.
 10. The pharmaceuticalcomposition of claim 9, wherein the composition is a powder.
 11. Thepharmaceutical composition of claim 9, wherein the composition is atablet.
 12. The pharmaceutical composition of claim 9, wherein thecomposition is granules.
 13. The pharmaceutical composition of claim 9,wherein the composition is a capsule.
 14. The pharmaceutical compositionof claim 8, in liquid form.
 15. The pharmaceutical composition of claim14, wherein the composition is a suspension.
 16. The pharmaceuticalcomposition of claim 15, wherein the composition is aqueous.
 17. Asingle unit dosage form comprising a compound of the formula:

or a pharmaceutically acceptable salt, solvate or stereoisomer thereofwherein: Z is substituted aryl, substituted or unsubstituted cycloalkyl,substituted alkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted arylalkyl; R¹ is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, —(CH₂CH₂O)_(n)R⁶ or any biohydrolyzable group; R²,R³, R⁴, R⁵ and R⁶ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl; substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, alkoxy, aryloxy, halogen, CF₃, OCF₃,OCHF₂, CN, COOH, COOR⁷, SO₂R⁷, NO₂, NH₂, or N(R⁷) ₂; each occurrence ofR⁷ is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl; substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, alkoxy, aryloxy, halogen or CF₃; n is an integerfrom 1 to 7, with the proviso that when R¹, R², R³, R⁴ and R⁵ are eachhydrogen, then Z is not 2-carboxy ethyl, 3-(4-pyridinyl)propyl,2-(4-piperidinyl) ethyl, 4-cyano-phenyl or alkyl substituted withsubstituted carbamyl, and a pharmaceutically acceptable carrier,excipient or diluent.
 18. A single unit dosage form comprising acompound of the formula:

or a pharmaceutically acceptable salt, solvate or stereoisomer thereofwherein: X is halogen, substituted alkyl, alkoxy or hydroxy; R¹ ishydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, —(CH₂CH₂O)_(n)R⁶ or any biohydrolyzable group; R², R³, R⁴,R⁵ and R⁶ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl; substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy,aryloxy, heteroaryloxy, halogen, CF₃, OCF₃, OCHF₂, CN, COOH, COOR⁷,SO₂R⁷, NO₂, NH₂, or N(R⁷)₂; each occurrence of R⁷ is independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl; substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, alkoxy, aryloxy, heteroaryloxy, halogen orCF₃; and n is an integer from 1 to 7, and a pharmaceutically acceptablecarrier, excipient or diluent.
 19. A single unit dosage form comprisinga compound selected from:3-[5-(4-Chloromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3,4-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Ethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Chloro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-tert-Butyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,5-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,4-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Chloro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Methoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,6-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Dimethylamino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Dimethylamino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3,5-Bis-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid; 3-[5-(3,4-Dimethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Pentyloxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3,4,5-Trimethoxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Isobutyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2,3-Difluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-5-methyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;4-Fluoro-3-[5-(4-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;2-Fluoro-5-[5-(4-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Chloro-2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Bromo-2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid sodium salt;3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester;5-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-2-methoxy-benzoic acid;3-[5-(2-Fluoro-6-hydroxy-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-methoxy-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-methoxy-ethoxy)-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy} -ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl ester;3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid2-[2-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethylester; 3-[5-(4-Amino-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Azido-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Benzyloxyg-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-p-Tolyl-[-1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(o-tolyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-m-Tolyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclohexyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclopropyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclopentyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Cyclobutyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Thiophen-2-yl-methyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(4-Chloro-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Chloro-phenoxymethyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-Benzyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-(5-Methoxymethyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(1-Phenyl-propyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(4-Fluoro-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Chloro-phenoxymethyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-Cyclopentylmethyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(4-Methoxy-benzyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-Biphenyl-4-yl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(4-Nitro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-(5-Propenyl-[1,2,4]oxadiazol-3-yl)-benzoic acid;3-[5-(4-Pyrrol-1-yl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Fluoro-biphenyl-4-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid;3-[5-(3-Fluoro-biphenyl-4-yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid; and apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, excipient or diluent.
 20. The single unit dosageform of claim 19, wherein the compound is3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof.
 21. The pharmaceuticalcomposition of claim 6, wherein the compound is3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid.
 22. Thesingle unit dosage form of claim 19, wherein the compound is3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid.